Oligomer conjugates of heteropentacyclic nucleosides

ABSTRACT

The invention provides small molecule drugs that are chemically modified by covalent attachment of a water-soluble oligomer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation Application of U.S. application Ser.No. 12/918,780, filed 27 Oct. 2010, now pending, which is a 35 U.S.C.§371 application of International Application No. PCT/US2009/001104,filed 20 Feb. 2009, designating the United States, which claims thebenefit of priority under 35 U.S.C. §119(e) to U.S. Provisional PatentApplication Ser. No. 61/066,815, filed 22 Feb. 2008, each of which areincorporated by reference in their entireties.

FIELD OF THE INVENTION

This invention comprises (among other things) chemically modifiedheteropentacyclic nucleosides that possess certain advantages overversions lacking the chemical modification. The chemically modifiedversions described herein relate to and/or have application(s) in (amongothers) the fields of drug discovery, pharmacotherapy, physiology,organic chemistry and polymer chemistry.

BACKGROUND OF THE INVENTION

Heteropentacyclic nucleosides (e.g., ribavirin, showdomycin andpyrazomycin) are agents having known pharmacologic activities. Theheteropentacyclic nucleoside ribavirin, for example, can inhibit thereplication of both RNA and DNA viruses. Showdomycin and pyrazomycin areheteropentacyclic nucleosides known to have antibiotic activity.

Some drugs in the heteropentacyclic nucleoside class are extensivelymetabolized via first-pass hepatic metabolism. In addition, some drugsin this class (and ribavirin in particular) can cause hemolytic anemia,a serious and often life-threatening condition. Anemia can occur whenribavirin diffuses into and accumulates within erythrocytes, due to aninability of erythrocytes to hydrolyze (dephosphorylate) the drug.Erythrocytes are unable to hydrolyze the drug because they lackphosphatases with the result that the ribavirin phosphate concentrationcan reach a level that is 60- to 100-fold higher than its plasmaconcentration. At these high levels, ribavirin phosphate can depleteintracellular ATP, impair ATP-dependent oxidative respiratory pathway,damage erythrocyte membrane integrity and eventually cause hemolyticanemia. Although these effects are reversible and may be mitigated byremoving the drug, reducing the drug or administering erythropoietin, itwould be advantageous to be able to administer a compound havingribavirin and other heteropentacyclic nucleosides without regard forthese or other undesired effects.

The present invention seeks to address these and other needs in the art.

SUMMARY OF THE INVENTION

In one or more embodiments of the invention, a compound is provided, thecompound comprising a heteropentacyclic nucleoside residue covalentlyattached via a stable or degradable linkage to a water-soluble,non-peptidic oligomer.

Exemplary compounds of the invention include those having the followingstructure:

wherein:

(HPC) is a five-membered heterocyclic moiety, preferably unsaturated andpreferably containing from one to three nitrogen atoms, more preferablyselected from the group consisting of

wherein R¹ is —C≡N, —C(O)NH₂, —C(S)NH₂, —C(O)OCH₃, —C(NH)NH₂ and—N(NH)N(H)OH;

R² is hydrogen or hydroxyl;

R³ is hydroxyl when R² is hydrogen and R³ is hydrogen when R² ishydroxyl;

X is a spacer moiety; and

POLY is a water-soluble, non-peptidic oligomer,

and 5′-phosphates and 3′,5′-cyclic phosphates thereof and ammonium andalkali metal salts of each of the 5′-phosphates and 3′,5′-cyclicphosphates.

Additional exemplary compounds of the invention include those having thefollowing structure:

wherein

(HPC)-X-[POLY]_(a) is a five-membered heterocyclic moiety, preferablyunsaturated and preferably containing from one to three nitrogen atomsto which attached is one (when a=1) or two (when a=2) POLY species (eachPOLY being a water-soluble, non-peptidic oligomer), through X, a spacermoiety, R² is hydrogen or hydroxyl; and R³ is hydroxyl when R² ishydrogen and R³ is hydrogen when R² is hydroxyl; and 5′-phosphates and3′,5′-cyclic phosphates thereof and ammonium and alkali metal salts ofeach of the 5′-phosphates and 3′,5′-cyclic phosphates. Preferably, priorto any attachment of a water-soluble, non-peptidic oligomer through thespacer moiety, the five-membered ring is preferably selected from thegroup consisting of

wherein R¹ is —C≡N, —C(O)NH₂, —C(S)NH₂, —C(O)OCH₃, —C(NH)NH₂ and—N(NH)N(H)OH.

Further exemplary compounds of the invention include those having thefollowing structure:

wherein:

R² is hydrogen or hydroxyl;

R³ is hydroxyl when R² is hydrogen and R³ is hydrogen when R² ishydroxyl;

X is a spacer moiety;

each POLY is independently a water-soluble, non-peptidic oligomer;

and (a) is one or two,

and 5′-phosphates and 3′,5′-cyclic phosphates thereof and ammonium andalkali metal salts of each of the 5′-phosphates and 3′,5′-cyclicphosphates.

Further exemplary compounds of the invention include those having thefollowing structure:

wherein:

R² is hydrogen or hydroxyl;

R³ is hydroxyl when R² is hydrogen and R³ is hydrogen when R² ishydroxyl;

each X is independently a spacer moiety;

each POLY is independently a water-soluble, non-peptidic oligomer;

and (a) is one or two,

and 5′-phosphates and 3′,5′-cyclic phosphates thereof and ammonium andalkali metal salts of each of the 5′-phosphates and 3′,5′-cyclicphosphates.

A “heteropentacyclic nucleoside residue” (or a “residue of aheteropentacyclic nucleoside”) is a compound having a structure of aheteropentacyclic nucleoside that is altered by the presence of one ormore bonds, which bonds serve to attach (either directly or indirectly)one or more water-soluble, non-peptidic oligomers. In this regard, anyheteropentacyclic nucleoside have pharmacologic activity can be used.Exemplary heteropentacyclic nucleosides have a structure encompassed bythe structure defined herein as Formula I, as follows:

wherein:

(HPC) is a five-membered heterocyclic moiety, preferably unsaturated andpreferably containing from one to three nitrogen atoms, more preferablyselected from the group consisting of

wherein R¹ is —C≡N, —C(O)NH₂, —C(S)NH₂, —C(O)OCH₃, —C(NH)NH₂ and—N(NH)N(H)OH;

R² is hydrogen or hydroxyl; and

R³ is hydroxyl when R² is hydrogen and R³ is hydrogen when R² ishydroxyl.

In one or more embodiments of the invention, a composition is provided,the composition comprising a compound comprising a residue of aheteropentacyclic nucleoside covalently attached via a stable ordegradable linkage to a water-soluble, non-peptidic oligomer, andoptionally, a pharmaceutically acceptable excipient.

In one or more embodiments of the invention, a dosage form is provided,the dosage form comprising a compound comprising a residue of aheteropentacyclic nucleoside covalently attached via a stable ordegradable linkage to a water-soluble, non-peptidic oligomer, whereinthe compound is present in a dosage form.

In one or more embodiments of the invention, a method is provided, themethod comprising covalently attaching a water-soluble, non-peptidicoligomer to a heteropentacyclic nucleoside.

In one or more embodiments of the invention, a method is provided, themethod comprising administering a compound comprising a residue of aheteropentacyclic nucleoside covalently attached via a stable ordegradable linkage to a water-soluble, non-peptidic oligomer.

These and other objects, aspects, embodiments and features of theinvention will become more fully apparent to one of ordinary skill inthe art when read in conjunction with the following detaileddescription.

DETAILED DESCRIPTION OF THE INVENTION

As used in this specification, the singular forms “a,” “an,” and “the”include plural referents unless the context clearly dictates otherwise.

In describing and claiming the present invention, the followingterminology will be used in accordance with the definitions describedbelow.

“Water soluble, non-peptidic oligomer” indicates an oligomer that is atleast 35% (by weight) soluble, preferably greater than 70% (by weight),and more preferably greater than 95% (by weight) soluble, in water atroom temperature. Typically, an unfiltered aqueous preparation of a“water-soluble” oligomer transmits at least 75%, more preferably atleast 95%, of the amount of light transmitted by the same solution afterfiltering. It is most preferred, however, that the water-solubleoligomer is at least 95% (by weight) soluble in water or completelysoluble in water. With respect to being “non-peptidic,” an oligomer isnon-peptidic when it has less than 35% (by weight) of amino acidresidues.

The terms “monomer,” “monomeric subunit” and “monomeric unit” are usedinterchangeably herein and refer to one of the basic structural units ofa polymer or oligomer. In the case of a homo-oligomer, a singlerepeating structural unit forms the oligomer. In the case of aco-oligomer, two or more structural units are repeated—either in apattern or randomly—to form the oligomer. Preferred oligomers used inconnection with present the invention are homo-oligomers. Thewater-soluble, non-peptidic oligomer typically comprises one or moremonomers serially attached to form a chain of monomers. The oligomer canbe formed from a single monomer type (i.e., is homo-oligomeric) or twoor three monomer types (i.e., is co-oligomeric).

An “oligomer” is a molecule possessing from about 1 to about 30monomers. Specific oligomers for use in the invention include thosehaving a variety of geometries such as linear, branched, or forked, tobe described in greater detail below.

“PEG” or “polyethylene glycol,” as used herein, is meant to encompassany water-soluble poly(ethylene oxide). Unless otherwise indicated, a“PEG oligomer” or an oligoethylene glycol is one in which substantiallyall (preferably all) monomeric subunits are ethylene oxide subunits,though the oligomer may contain distinct end capping moieties orfunctional groups, e.g., for conjugation. PEG oligomers for use in thepresent invention may comprise the following structures:“—(CH₂CH₂O)_(n)—” or “—(CH₂CH₂O)_(n-1)CH₂CH₂—,” depending upon whetheror not the terminal oxygen(s) has been displaced, e.g., during asynthetic transformation. As stated above, for the PEG oligomers, thevariable (n) ranges from 1 to 30, and the terminal groups andarchitecture of the overall PEG can vary. When PEG further comprises afunctional group, A, for linking to, e.g., a small molecule drug, thefunctional group when covalently attached to a PEG oligomer does notresult in formation of (i) an oxygen-oxygen bond (—O—O—, a peroxidelinkage), or (ii) a nitrogen-oxygen bond (N—O, O—N).

The terms “end-capped” or “terminally capped” are interchangeably usedherein to refer to a terminal or endpoint of a polymer having anend-capping moiety. Typically, although not necessarily, the end-cappingmoiety comprises a hydroxy or C₁₋₂₀ alkoxy group. Thus, examples ofend-capping moieties include alkoxy (e.g., methoxy, ethoxy andbenzyloxy), as well as aryl, heteroaryl, cyclo, heterocyclo, and thelike. In addition, saturated, unsaturated, substituted and unsubstitutedforms of each of the foregoing are envisioned. Moreover, the end-cappinggroup can also be a silane. The end-capping group can alsoadvantageously comprise a detectable label. When the polymer has anend-capping group comprising a detectable label, the amount or locationof the polymer and/or the moiety (e.g., active agent) of interest towhich the polymer is coupled, can be determined by using a suitabledetector. Such labels include, without limitation, fluorescers,chemiluminescers, moieties used in enzyme labeling, colorimetricmoieties (e.g., dyes), metal ions, radioactive moieties, and the like.Suitable detectors include photometers, films, spectrometers, and thelike.

“Branched,” in reference to the geometry or overall structure of anoligomer, refers to an oligomer having two or more polymer “arms”extending from a branch point.

“Forked,” in reference to the geometry or overall structure of anoligomer, refers to an oligomer having two or more functional groups(through one or more atoms) extending from a branch point.

A “branch point” refers to a bifurcation point comprising one or moreatoms at which an oligomer branches or forks from a linear structureinto one or more additional arms.

The term “reactive” or “activated” refers to a functional group thatreacts readily or at a practical rate under conventional conditions oforganic synthesis. This is in contrast to those groups that either donot react or require strong catalysts or impractical reaction conditionsin order to react (i.e., a “nonreactive” or “inert” group).

“Not readily reactive,” with reference to a functional group present ona molecule in a reaction mixture, indicates that the group remainslargely intact under conditions that are effective to produce a desiredreaction in the reaction mixture.

A “protecting group” is a moiety that prevents or blocks reaction of aparticular chemically reactive functional group in a molecule undercertain reaction conditions. The protecting group may vary dependingupon the type of chemically reactive group being protected as well asthe reaction conditions to be employed and the presence of additionalreactive or protecting groups in the molecule. Functional groups whichmay be protected include, by way of example, carboxylic acid groups,amino groups, hydroxyl groups, thiol groups, carbonyl groups and thelike. Representative protecting groups for carboxylic acids includeesters (such as a p-methoxybenzyl ester), amides and hydrazides; foramino groups, carbamates (such as tert-butoxycarbonyl) and amides; forhydroxyl groups, ethers and esters; for thiol groups, thioethers andthioesters; for carbonyl groups, acetals and ketals; and the like. Suchprotecting groups are well-known to those skilled in the art and aredescribed, for example, in T. W. Greene and G. M. Wuts, ProtectingGroups in Organic Synthesis, Third Edition, Wiley, New York, 1999, andreferences cited therein.

A functional group in “protected form” refers to a functional groupbearing a protecting group. As used herein, the term “functional group”or any synonym thereof encompasses protected forms thereof.

A “physiologically cleavable” or “hydrolyzable” or “degradable” bond isa relatively labile bond that reacts with water (i.e., is hydrolyzed)under physiological conditions. The tendency of a bond to hydrolyze inwater may depend not only on the general type of linkage connecting twocentral atoms but also on the substituents attached to these centralatoms. Appropriate hydrolytically unstable or weak linkages include butare not limited to carboxylate ester, phosphate ester, anhydrides,acetals, ketals, acyloxyalkyl ether, imines, orthoesters, peptides,oligonucleotides, thioesters, thiolesters, and carbonates.

An “enzymatically degradable linkage” means a linkage that is subject todegradation by one or more enzymes.

A “stable” linkage or bond refers to a chemical bond that issubstantially stable in water, that is to say, does not undergohydrolysis under physiological conditions to any appreciable extent overan extended period of time. Examples of hydrolytically stable linkagesinclude but are not limited to the following: carbon-carbon bonds (e.g.,in aliphatic chains), ethers, amides, urethanes, amines, and the like.Generally, a stable linkage is one that exhibits a rate of hydrolysis ofless than about 1-2% per day under physiological conditions. Hydrolysisrates of representative chemical bonds can be found in most standardchemistry textbooks.

“Substantially” or “essentially” means nearly totally or completely, forinstance, 95% or greater, more preferably 97% or greater, still morepreferably 98% or greater, even more preferably 99% or greater, yetstill more preferably 99.9% or greater, with 99.99% or greater beingmost preferred of some given quantity.

“Monodisperse” refers to an oligomer composition wherein substantiallyall of the oligomers in the composition have a well-defined, single(i.e., the same) molecular weight and defined number of monomers, asdetermined by chromatography or mass spectrometry. Monodisperse oligomercompositions are in one sense pure, that is, substantially having asingle and definable number (as a whole number) of monomers rather thana large distribution. A monodisperse oligomer composition possesses aMW/Mn value of 1.0005 or less, and more preferably, a MW/Mn value of1.0000. By extension, a composition comprised of monodisperse conjugatesmeans that substantially all oligomers of all conjugates in thecomposition have a single and definable number (as a whole number) ofmonomers rather than a large distribution and would possess a MW/Mnvalue of 1.0005, and more preferably, a MW/Mn value of 1.0000 if theoligomer were not attached to the corticosteroid residue. A compositioncomprised of monodisperse conjugates may, however, include one or morenonconjugate substances such as solvents, reagents, excipients, and soforth.

“Bimodal,” in reference to an oligomer composition, refers to anoligomer composition wherein substantially all oligomers in thecomposition have one of two definable and different numbers (as wholenumbers) of monomers rather than a large distribution, and whosedistribution of molecular weights, when plotted as a number fractionversus molecular weight, appears as two separate identifiable peaks.Preferably, for a bimodal oligomer composition as described herein, eachpeak is generally symmetric about its mean, although the size of the twopeaks may differ. Ideally, the polydispersity index of each peak in thebimodal distribution, Mw/Mn, is 1.01 or less, more preferably 1.001 orless, and even more preferably 1.0005 or less, and most preferably aMW/Mn value of 1.0000. By extension, a composition comprised of bimodalconjugates means that substantially all oligomers of all conjugates inthe composition have one of two definable and different numbers (aswhole numbers) of monomers rather than a large distribution and wouldpossess a MW/Mn value of 1.01 or less, more preferably 1.001 or less andeven more preferably 1.0005 or less, and most preferably a MW/Mn valueof 1.0000 if the oligomer were not attached to the corticosteroidresidue. A composition comprised of bimodal conjugates may, however,include one or more nonconjugate substances such as solvents, reagents,excipients, and so forth

A “heteropentacyclic nucleoside” refers to an organic, inorganic, ororganometallic compound having a molecular weight of less than about1000 Daltons (and typically less than 500 Daltons) and having somepharmacological activity, such as antiviral and/or antibiotic activity.

A “biological membrane” is any membrane made of cells or tissues thatserves as a barrier to at least some foreign entities or otherwiseundesirable materials. As used herein a “biological membrane” includesthose membranes that are associated with physiological protectivebarriers including, for example: the blood-brain barrier (BBB); theblood-cerebrospinal fluid barrier; the blood-placental barrier; theblood-milk barrier; the blood-testes barrier; and mucosal barriersincluding the vaginal mucosa, urethral mucosa, anal mucosa, buccalmucosa, sublingual mucosa, and rectal mucosa. Unless the context clearlydictates otherwise, the term “biological membrane” does not includethose membranes associated with the middle gastro-intestinal tract(e.g., stomach and small intestines).

A “biological membrane crossing rate,” provides a measure of acompound's ability to cross a biological membrane, such as theblood-brain barrier (“BBB”). A variety of methods may be used to assesstransport of a molecule across any given biological membrane. Methods toassess the biological membrane crossing rate associated with any givenbiological barrier (e.g., the blood-cerebrospinal fluid barrier, theblood-placental barrier, the blood-milk barrier, the intestinal barrier,and so forth), are known, described herein and/or in the relevantliterature, and/or may be determined by one of ordinary skill in theart.

A “reduced rate of metabolism” refers to a measurable reduction in therate of metabolism of a water-soluble oligomer-small molecule drugconjugate as compared to the rate of metabolism of the small moleculedrug not attached to the water-soluble oligomer (i.e., the smallmolecule drug itself) or a reference standard material. In the specialcase of “reduced first pass rate of metabolism,” the same “reduced rateof metabolism” is required except that the small molecule drug (orreference standard material) and the corresponding conjugate areadministered orally. Orally administered drugs are absorbed from thegastro-intestinal tract into the portal circulation and may pass throughthe liver prior to reaching the systemic circulation. Because the liveris the primary site of drug metabolism or biotransformation, asubstantial amount of drug may be metabolized before it reaches thesystemic circulation. The degree of first pass metabolism, and thus, anyreduction thereof, may be measured by a number of different approaches.For instance, animal blood samples may be collected at timed intervalsand the plasma or serum analyzed by liquid chromatography/massspectrometry for metabolite levels. Other techniques for measuring a“reduced rate of metabolism” associated with the first pass metabolismand other metabolic processes are known, described herein and/or in therelevant literature, and/or can be determined by one of ordinary skillin the art. Preferably, a conjugate of the invention can provide areduced rate of metabolism reduction satisfying at least one of thefollowing values: at least about 5%; at least about 15%; at least about20%; at least about 25%; at least about 30%; at least about 40%; atleast about 60%, at least about 70%, at least about 80%, and at leastabout 90%.

“Alkyl” refers to a hydrocarbon chain, ranging from about 1 to 20 atomsin length. Such hydrocarbon chains are preferably but not necessarilysaturated and may be branched or straight chain. Exemplary alkyl groupsinclude methyl, ethyl, propyl, butyl, pentyl, 2-methylbutyl,2-ethylpropyl, 3-methylpentyl, and the like. As used herein, “alkyl”includes cycloalkyl when three or more carbon atoms are referenced.

“Lower alkyl” refers to an alkyl group containing from 1 to 6 carbonatoms, and may be straight chain or branched, as exemplified by methyl,ethyl, n-butyl, i-butyl, t-butyl.

“Non-interfering substituents” are those groups that, when present in amolecule, are typically non-reactive with other functional groupscontained within the molecule.

“Alkoxy” refers to an —O—R group, wherein R is alkyl or substitutedalkyl, preferably C₁-C₂₀ alkyl (e.g., methoxy, ethoxy, propyloxy,benzyl, etc.), preferably C₁-C₇.

“Pharmaceutically acceptable excipient” or “pharmaceutically acceptablecarrier” refers to component that may be included in the compositions ofthe invention and that causes no significant adverse toxicologicaleffects to a patient.

The term “aryl” means an aromatic group having up to 14 carbon atoms.Aryl groups include phenyl, naphthyl, biphenyl, phenanthrenyl,naphthacenyl, and the like. “Substituted phenyl” and “substituted aryl”denote a phenyl group and aryl group, respectively, substituted withone, two, three, four or five (e.g. 1-2, 1-3 or 1-4 substituents) chosenfrom halo (F, Cl, Br, l), hydroxy, hydroxy, cyano, nitro, alkyl (e.g.,C₁₋₆ alkyl), alkoxy (e.g., C₁₋₆ alkoxy), benzyloxy, carboxy, aryl, andso forth.

For simplicity, chemical moieties are defined and referred to throughoutprimarily as univalent chemical moieties (e.g., alkyl, aryl, etc.),divalent moieties (e.g., a spacer moiety providing a “bridge” betweentwo moieties), or polyvalent moieties (e.g., a spacer moiety providing a“bridge” between three or more moieties). For any given moiety, one ofordinary skill in the art will be able to understand the requiredvalence based on the chemical structures provided herein. For moietiesthat are individual atoms, all atoms are understood to have their normalnumber of valences for bond formation (i.e., 4 for carbon, 3 for N, 2for O, and 2, 4, or 6 for S, depending on the oxidation state of the S).

“Pharmacologically effective amount,” “physiologically effectiveamount,” and “therapeutically effective amount” are used interchangeablyherein to mean the amount of a water-soluble oligomer-small moleculedrug conjugate present in a composition that is needed to provide adesired level of active agent and/or conjugate in the bloodstream or inthe target tissue. The precise amount may depend upon numerous factors,e.g., the particular active agent, the components and physicalcharacteristics of the composition, intended patient population, patientconsiderations, and may readily be determined by one skilled in the art,based upon the information provided herein and available in the relevantliterature.

A “difunctional” oligomer is an oligomer having two functional groupscontained therein, typically at its termini. When the functional groupsare the same, the oligomer is said to be homodifunctional. When thefunctional groups are different, the oligomer is said to beheterobifunctional.

A basic reactant or an acidic reactant described herein include neutral,charged, and any corresponding salt forms thereof.

The term “patient,” refers to a living organism suffering from or proneto a condition that can be prevented or treated by administration of aconjugate as described herein.

“Optional” or “optionally” means that the subsequently describedcircumstance may but need not necessarily occur, so that the descriptionincludes instances where the circumstance occurs and instances where itdoes not.

As indicated above, the present invention is directed to (among otherthings) a compound comprising a residue of heteropentacyclic nucleosidecovalently attached via a stable or degradable linkage to awater-soluble, non-peptidic oligomer.

In one or more embodiments of the invention, a compound is provided, thecompound comprising a residue of heteropentacyclic nucleoside covalentlyattached via a stable or degradable linkage to a water-soluble,non-peptidic oligomer, wherein the heteropentacyclic nucleoside has astructure encompassed by the following formula:

wherein:

(HPC) is a five-membered heterocyclic moiety, preferably unsaturated andpreferably containing from one to three nitrogen atoms, more preferablyselected from the group consisting of

wherein R¹ is —C≡N, —C(O)NH₂, —C(S)NH₂, —C(O)OCH₃, —C(NH)NH₂ and—N(NH)N(H)OH;

R² is hydrogen or hydroxyl; and

R³ is hydroxyl when R² is hydrogen and R³ is hydrogen when R² ishydroxyl.

The small molecule to which the water-soluble, nonpeptidic oligomer isattached can be a heteropentacyclic nucleoside. Specific examples ofwhich include ribavirin, showdomycin, and pyrazomycin.

It is believed that an advantage of the compounds of the presentinvention is their ability to retain some degree of pharmacologicactivity while also exhibiting a decrease in side effects uponadministration to a patient. Although not wishing to be bound by theory,it is believed that the oligomer-containing compounds describedherein—in contrast to the oligomer-free “original” heteropentacyclicnucleoside—are not metabolized as readily because the oligomer serves toreduce the overall affinity of the compound to substrates that canmetabolize heterocyclic nucleosides. Further, it is believed that thecompounds of the present invention have reduced uptake and subsequentaccumulation in erythrocytes as compared to the oligomer-free version ofthe heteropentacyclic nucleoside. Even should the linkage between theresidue of the heteropentacyclic nucleoside and the oligomer bereleasable, the compound still offers advantages (such as avoidingfirst-pass metabolism upon initial absorption).

As indicated above, the compounds of the invention include a residue ofa heteropentacyclic nucleoside. Assays for determining whether a givencompound (regardless of whether the compound includes a water-soluble,non-peptidic oligomer or not) has activity are described herein and/orare otherwise known to those of ordinary skill in the art.

Exemplary heteropentacyclic nucleosides are encompassed by the followingformula:

wherein:

(HPC) is a five-membered heterocyclic moiety, preferably unsaturated andpreferably containing from one to three nitrogen atoms, more preferablyselected from the group consisting of

wherein R¹ is —C≡N, —C(O)NH₂, —C(S)NH₂, —C(O)OCH₃, —C(NH)NH₂ and—N(NH)N(H)OH;

R² is hydrogen or hydroxyl; and

R³ is hydroxyl when R² is hydrogen and R³ is hydrogen when R² ishydroxyl.

In one or more embodiments, the heteropentacyclic nucleoside isencompassed by the following structure:

wherein: R¹ is —C≡N, —C(O)NH₂, —C(S)NH₂, —C(O)OCH₃, —C(NH)NH₂ and—N(NH)N(H)OH; R² is hydrogen or hydroxyl; and R³ is hydroxyl when R² ishydrogen and R³ is hydrogen when R² is hydroxyl. In one or moreembodiments, the small molecule is ribavirin, which has the followingstructure:

Compounds encompassed by Formula Ia are described in U.S. Pat. No.3,798,209 and can be prepared according to known methods.

In some instances, heteropentacyclic nucleosides can be obtained fromcommercial sources. In addition, heteropentacyclic nucleosides can beobtained through chemical synthesis. Examples of heteropentacyclicnucleosides as well as synthetic approaches for preparing the same aredescribed in the literature and in, for example, U.S. Pat. No.3,798,209.

Each of these (and other) heteropentacyclic nucleosides can becovalently attached (either directly or through one or more atoms) to awater-soluble, non-peptidic oligomer.

Small molecule drugs useful in the invention generally have a molecularweight of less than 1000 Da. Exemplary molecular weights of smallmolecule drugs include molecular weights of: less than about 950; lessthan about 900; less than about 850; less than about 800; less thanabout 750; less than about 700; less than about 650; less than about600; less than about 550; less than about 500; less than about 450; lessthan about 400; less than about 350; and less than about 300.

The small molecule drug used in the invention, if chiral, may be in aracemic mixture, or an optically active form, for example, a singleoptically active enantiomer, or any combination or ratio of enantiomers(i.e., scalemic mixture). In addition, the small molecule drug maypossess one or more geometric isomers. With respect to geometricisomers, a composition can comprise a single geometric isomer or amixture of two or more geometric isomers. A small molecule drug for usein the present invention can be in its customary active form, or maypossess some degree of modification. For example, a small molecule drugmay have a targeting agent, tag, or transporter attached thereto, priorto or after covalent attachment of an oligomer. Alternatively, the smallmolecule drug may possess a lipophilic moiety attached thereto, such asa phospholipid (e.g., distearoylphosphatidylethanolamine or “DSPE,”dipalmitoylphosphatidylethanolarnine or “DPPE,” and so forth) or a smallfatty acid. In some instances, however, it is preferred that the smallmolecule drug moiety does not include attachment to a lipophilic moiety.

Heteropentacyclic nucleosides for coupling to a water-soluble,non-peptidic oligomer possesses a free hydroxyl, carboxyl, thio, aminogroup, or the like (i.e., “handle”) suitable for covalent attachment tothe oligomer. In addition, a heteropentacyclic nucleoside can bemodified by introduction of a reactive group, preferably by conversionof one of its existing functional groups to a functional group suitablefor formation of a stable covalent linkage between the oligomer and thedrug. Both approaches are illustrated in the Experimental section.

Accordingly, each oligomer is composed of up to three different monomertypes selected from the group consisting of: alkylene oxide, such asethylene oxide or propylene oxide; olefinic alcohol, such as vinylalcohol, 1-propenol or 2-propenol; vinyl pyrrolidone; hydroxyalkylmethacrylamide or hydroxyalkyl methacrylate, where alkyl is preferablymethyl; α-hydroxy acid, such as lactic acid or glycolic acid;phosphazene, oxazoline, amino acids, carbohydrates such asmonosaccharides, saccharide or mannitol; and N-acryloylmorpholine.Preferred monomer types include alkylene oxide, olefinic alcohol,hydroxyalkyl methacrylamide or methacrylate, N-acryloylmorpholine, andα-hydroxy acid. Preferably, each oligomer is, independently, aco-oligomer of two monomer types selected from this group, or, morepreferably, is a homo-oligomer of one monomer type selected from thisgroup.

The two monomer types in a co-oligomer may be of the same monomer type,for example, two alkylene oxides, such as ethylene oxide and propyleneoxide. Preferably, the oligomer is a homo-oligomer of ethylene oxide.Usually, although not necessarily, the terminus (or termini) of theoligomer that is not covalently attached to a small molecule is cappedto render it unreactive. Alternatively, the terminus may include areactive group. When the terminus is a reactive group, the reactivegroup is either selected such that it is unreactive under the conditionsof formation of the final oligomer or during covalent attachment of theoligomer to a small molecule drug, or it is protected as necessary. Onecommon end-functional group is hydroxyl or —OH, particularly foroligoethylene oxides.

The water-soluble, non-peptidic oligomer (e.g., “POLY” in variousstructures provided herein) can have any of a number of differentgeometries. For example, the water-soluble, non-peptidic oligomer it canbe linear, branched, or forked. Most typically, the water-soluble,non-peptidic oligomer is linear or is branched, for example, having onebranch point. Although much of the discussion herein is focused uponpoly(ethylene oxide) as an illustrative oligomer, the discussion andstructures presented herein can be readily extended to encompass anywater-soluble, non-peptidic oligomers described above.

The molecular weight of the water-soluble, non-peptidic oligomer,excluding the linker portion, is generally relatively low. Exemplaryvalues of the molecular weight of the water-soluble polymer include:below about 1500; below about 1450; below about 1400; below about 1350;below about 1300; below about 1250; below about 1200; below about 1150;below about 1100; below about 1050; below about 1000; below about 950;below about 900; below about 850; below about 800; below about 750;below about 700; below about 650; below about 600; below about 550;below about 500; below about 450; below about 400; below about 350;below about 300; below about 250; below about 200; and below about 100Daltons.

Exemplary ranges of molecular weights of the water-soluble, non-peptidicoligomer (excluding the linker) include: from about 100 to about 1400Daltons; from about 100 to about 1200 Daltons; from about 100 to about800 Daltons; from about 100 to about 500 Daltons; from about 100 toabout 400 Daltons; from about 200 to about 500 Daltons; from about 200to about 400 Daltons; from about 75 to 1000 Daltons; and from about 75to about 750 Daltons.

Preferably, the number of monomers in the water-soluble, non-peptidicoligomer falls within one or more of the following ranges: between about1 and about 30 (inclusive); between about 1 and about 25; between about1 and about 20; between about 1 and about 15; between about 1 and about12; between about 1 and about 10. In certain instances, the number ofmonomers in series in the oligomer (and the corresponding conjugate) isone of 1, 2, 3, 4, 5, 6, 7, or 8. In additional embodiments, theoligomer (and the corresponding conjugate) contains 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 monomers. In yet further embodiments, theoligomer (and the corresponding conjugate) possesses 21, 22, 23, 24, 25,26, 27, 28, 29 or 30 monomers in series. Thus, for example, when thewater-soluble, non-peptidic polymer includes CH₃—(OCH₂CH₂)_(n)—, “n” isan integer that can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, andcan fall within one or more of the following ranges: between about 1 andabout 25; between about 1 and about 20; between about 1 and about 15;between about 1 and about 12; between about 1 and about 10.

When the water-soluble, non-peptidic oligomer has 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 monomers, these values correspond to a methoxy end-cappedoligo(ethylene oxide) having a molecular weights of about 75, 119, 163,207, 251, 295, 339, 383, 427, and 471 Daltons, respectively. When theoligomer has 11, 12, 13, 14, or 15 monomers, these values correspond tomethoxy end-capped oligo(ethylene oxide) having molecular weightscorresponding to about 515, 559, 603, 647, and 691 Daltons,respectively.

When the water-soluble, non-peptidic oligomer is attached to theheteropentacyclic nucleoside (in contrast to the step-wise addition ofone or more monomers to effectively “grow” the oligomer onto theheteropentacyclic nucleoside), it is preferred that the compositioncontaining an activated form of the water-soluble, non-peptidic oligomerbe monodisperse. In those instances, however, where a bimodalcomposition is employed, the composition will possess a bimodaldistribution centering around any two of the above numbers of monomers.For instance, a bimodal oligomer may have any one of the followingexemplary combinations of monomer subunits: 1-2, 1-3, 1-4, 1-5, 1-6,1-7, 1-8, 1-9, 1-10, and so forth; 2-3, 2-4, 2-5, 2-6, 2-7, 2-8, 2-9,2-10, and so forth; 3-4, 3-5, 3-6, 3-7, 3-8, 3-9, 3-10, and so forth;4-5, 4-6, 4-7, 4-8, 4-9, 4-10, and so forth; 5-6, 5-7, 5-8, 5-9, 5-10,and so forth; 6-7, 6-8, 6-9, 6-10, and so forth; 7-8, 7-9, 7-10, and soforth; and 8-9, 8-10, and so forth.

In some instances, the composition containing an activated form of thewater-soluble, non-peptidic oligomer will be trimodal or eventetramodal, possessing a range of monomers units as previouslydescribed. Oligomer compositions possessing a well-defined mixture ofoligomers (i.e., being bimodal, trimodal, tetramodal, and so forth) canbe prepared by mixing purified monodisperse oligomers to obtain adesired profile of oligomers (a mixture of two oligomers differing onlyin the number of monomers is bimodal; a mixture of three oligomersdiffering only in the number of monomers is trimodal; a mixture of fouroligomers differing only in the number of monomers is tetramodal), oralternatively, can be obtained from column chromatography of apolydisperse oligomer by recovering the “center cut,” to obtain amixture of oligomers in a desired and defined molecular weight range.

It is preferred that the water-soluble, non-peptidic oligomer isobtained from a composition that is preferably unimolecular ormonodisperse. That is, the oligomers in the composition possess the samediscrete molecular weight value rather than a distribution of molecularweights. Some monodisperse oligomers can be purchased from commercialsources such as those available from Sigma-Aldrich, or alternatively,can be prepared directly from commercially available starting materialssuch as Sigma-Aldrich. Water-soluble, non-peptidic oligomers can beprepared as described in Chen Y, Baker, G. L., J. Org. Chem., 6870-6873(1999), WO 02/098949, and U.S. Patent Application Publication2005/0136031.

When present, the spacer moiety (through which the water-soluble,non-peptidic polymer is attached to the heteropentacyclic nucleoside)may be a single bond, a single atom, such as an oxygen atom or a sulfuratom, two atoms, or a number of atoms. A spacer moiety is typically butis not necessarily linear in nature. The spacer moiety, “X,” ishydrolytically stable, and is preferably also enzymatically stable.Preferably, the spacer moiety “X” is one having a chain length of lessthan about 12 atoms, and preferably less than about 10 atoms, and evenmore preferably less than about 8 atoms and even more preferably lessthan about 5 atoms, whereby length is meant the number of atoms in asingle chain, not counting substituents. For instance, a urea linkagesuch as this, R_(oligomer)—NH—(C═O)—NH—R′_(drug), is considered to havea chain length of 3 atoms (—NH—C(O)—NH—). In selected embodiments, thelinkage does not comprise further spacer groups.

In some instances, the spacer moiety “X” comprises an ether, amide,urethane, amine, thioether, urea, or a carbon-carbon bond. Functionalgroups such as those discussed below, and illustrated in the examples,are typically used for forming the linkages. The spacer moiety may lesspreferably also comprise (or be adjacent to or flanked by) other atoms,as described further below.

More specifically, in selected embodiments, a spacer moiety of theinvention, X, may be any of the following: “—” (i.e., a covalent bond,that may be stable or degradable, between the heteropentacyclicnucleoside and the water-soluble, non-peptidic oligomer), —C(NH)NH₂—,—NH₂C(NH)—, —O—, —NH—, —S—, —C(O)—, C(O)—NH, NH—C(O)—NH, —O—C(O)—NH,—C(S)—, —CH₂—, —CH₂—CH₂—, —CH₂—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—, —O—CH₂—,—CH₂—O—, —O—CH₂—CH₂—, —CH₂—O—CH₂—, —CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—, —CH₂—CH₂—CH₂—O—, —O—CH₂—CH₂—CH₂—CH₂—,—CH₂—O—CH₂—CH₂—CH₂—, —CH₂—CH₂—O—CH₂—CH₂—, —CH₂—CH₂—CH₂—O—CH₂—,—CH₂—CH₂—CH₂—CH₂—O—, —C(O)—NH—CH₂—, —C(O)—NH—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—C(O)—NH—, —C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—C(O)—NH—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—,—C(O)—NH—CH₂—CH₂—CH₂—CH₂—, —CH₂—C(O)—NH—CH₂—CH₂—CH₂—,—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—, —CH₂—CH₂—CH₂—CH₂—C(O)—NH—NH—C(O)—CH₂—,—CH₂—NH—C(O)—CH₂—, —CH₂—CH₂—NH—C(O)—CH₂—, —NH—C(O)—CH₂—CH₂—,—CH₂—NH—C(O)—CH₂—CH₂, —CH₂—CH₂—NH—C(O)—CH₂—CH₂, —C(O)—NH—CH₂—,—C(O)—NH—CH₂—CH₂—, —O—C(O)—NH—CH₂—, —O—C(O)—NH—CH₂—CH₂—, —NH—CH₂—,—NH—CH₂—CH₂—, —CH₂—NH—CH₂—, —CH₂—CH₂—NH—CH₂—, —C(O)—CH₂—,—C(O)—CH₂—CH₂—, —CH₂—C(O)—CH₂—, —CH₂—CH₂—C(O)—CH₂—,—CH₂—CH₂—C(O)—CH₂—CH₂—, —CH₂—CH₂—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—,—CH₂—CH₂—CH₂—C(O)—NH—CH₂—CH₂—NH—C(O)—CH₂—, bivalent cycloalkyl group,—N(R⁶)—, R⁶ is H or an organic radical selected from the groupconsisting of alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkynyl, substituted alkynyl, aryl and substituted aryl.

For purposes of the present invention, however, a group of atoms is notconsidered a linkage when it is immediately adjacent to an oligomersegment, and the group of atoms is the same as a monomer of the oligomersuch that the group would represent a mere extension of the oligomerchain.

The linkage “X” between the water-soluble, non-peptidic oligomer and thesmall molecule is typically formed by reaction of a functional group ona terminus of the oligomer (or nascent oligomer when it is desired to“grow” the oligomer onto the heteropentacyclic nucleoside) with acorresponding functional group within the heteropentacyclic nucleoside.Illustrative reactions are described briefly below. For example, anamino group on an oligomer may be reacted with a carboxylic acid or anactivated carboxylic acid derivative on the small molecule, or viceversa, to produce an amide linkage. Alternatively, reaction of an amineon an oligomer with an activated carbonate (e.g. succinimidyl orbenzotriazyl carbonate) on the drug, or vice versa, forms a carbamatelinkage. Reaction of an amine on an oligomer with an isocyanate(R—N═C═O) group on a drug, or vice versa, forms a urea linkage(R—NH—(C═O)—NH—R′). Further, reaction of an alcohol (alkoxide) group onan oligomer with an alkyl halide, or halide group within a drug, or viceversa, forms an ether linkage. In yet another coupling approach, a smallmolecule having an aldehyde group is coupled to an oligomer amino groupby reductive amination, resulting in formation of a secondary aminelinkage between the oligomer and the small molecule.

A particularly preferred water-soluble, non-peptidic oligomer is anoligomer bearing an aldehyde functional group. In this regard, theoligomer will have the following structure:CH₃O—(CH₂—CH₂—O)_(n)—(CH₂)_(p)—C(O)H, wherein (n) is one of 1, 2, 3, 4,5, 6, 7, 8, 9 and 10 and (p) is one of 1, 2, 3, 4, 5, 6 and 7. Preferred(n) values include 3, 5 and 7 and preferred (p) values 2, 3 and 4.

Optionally, the terminus of the water-soluble, non-peptidic oligomer notbearing a functional group is capped to render it unreactive. When theoligomer includes a further functional group at a terminus other thanthat intended for formation of a conjugate, that group is eitherselected such that it is unreactive under the conditions of formation ofthe linkage “X,” or it is protected during the formation of the linkage“X.”

As stated above, the water-soluble, non-peptidic oligomer includes atleast one functional group prior to conjugation. The functional grouptypically comprises an electrophilic or nucleophilic group for covalentattachment to a small molecule, depending upon the reactive groupcontained within or introduced into the small molecule. Examples ofnucleophilic groups that may be present in either the oligomer or thesmall molecule include hydroxyl, amine, hydrazine (—NHNH₂), hydrazide(—C(O)NHNH₂), and thiol. Preferred nucleophiles include amine,hydrazine, hydrazide, and thiol, particularly amine. Most small moleculedrugs for covalent attachment to an oligomer will possess a freehydroxyl, amino, thio, aldehyde, ketone, or carboxyl group.

Examples of electrophilic functional groups that may be present ineither the oligomer or the small molecule include carboxylic acid,carboxylic ester, particularly imide esters, orthoester, carbonate,isocyanate, isothiocyanate, aldehyde, ketone, thione, alkenyl, acrylate,methacrylate, acrylamide, sulfone, maleimide, disulfide, iodo, epoxy,sulfonate, thiosulfonate, silane, alkoxysilane, and halosilane. Morespecific examples of these groups include succinimidyl ester orcarbonate, imidazoyl ester or carbonate, benzotriazole ester orcarbonate, vinyl sulfone, chloroethylsulfone, vinylpyridine, pyridyldisulfide, iodoacetamide, glyoxal, dione, mesylate, tosylate, andtresylate (2,2,2-trifluoroethanesulfonate).

Also included are sulfur analogs of several of these groups, such asthione, thione hydrate, thioketal, 2-thiazolidine thione, etc., as wellas hydrates or protected derivatives of any of the above moieties (e.g.aldehyde hydrate, hemiacetal, acetal, ketone hydrate, hemiketal, ketal,thioketal, thioacetal).

An “activated derivative” of a carboxylic acid refers to a carboxylicacid derivative that reacts readily with nucleophiles, generally muchmore readily than the underivatized carboxylic acid. Activatedcarboxylic acids include, for example, acid halides (such as acidchlorides), anhydrides, carbonates, and esters. Such esters includeimide esters, of the general form —(CO)O—N[(CO)—]₂; for example,N-hydroxysuccinimidyl (NHS) esters or N-hydroxyphthalimidyl esters. Alsopreferred are imidazolyl esters and benzotriazole esters. Particularlypreferred are activated propionic acid or butanoic acid esters, asdescribed in co-owned U.S. Pat. No. 5,672,662. These include groups ofthe form —(CH₂)₂₋₃C(═O)O-Q, where Q is preferably selected fromN-succinimide, N-sulfosuccinimide, N-phthalimide, N-glutarimide,N-tetrahydrophthalimide, N-norbornene-2,3-dicarboximide, benzotriazole,7-azabenzotriazole, and imidazole.

Other preferred electrophilic groups include succinimidyl carbonate,maleimide, benzotriazole carbonate, glycidyl ether, imidazoyl carbonate,p-nitrophenyl carbonate, acrylate, tresylate, aldehyde, and orthopyridyldisulfide.

These electrophilic groups are subject to reaction with nucleophiles,e.g., hydroxy, thio, or amino groups, to produce various bond types.Preferred for the present invention are reactions which favor formationof a hydrolytically stable linkage. For example, carboxylic acids andactivated derivatives thereof, which include orthoesters, succinimidylesters, imidazolyl esters, and benzotriazole esters, react with theabove types of nucleophiles to form esters, thioesters, and amides,respectively, of which amides are the most hydrolytically stable.Carbonates, including succinimidyl, imidazolyl, and benzotriazolecarbonates, react with amino groups to form carbamates. Isocyanates(R—N═C═O) react with hydroxyl or amino groups to form, respectively,carbamate (RNH—C(O)—OR′) or urea (RNH—C(O)—NHR′) linkages. Aldehydes,ketones, glyoxals, diones and their hydrates or alcohol adducts (i.e.,aldehyde hydrate, hemiacetal, acetal, ketone hydrate, hemiketal, andketal) are preferably reacted with amines, followed by reduction of theresulting imine, if desired, to provide an amine linkage (reductiveamination).

Several of the electrophilic functional groups include electrophilicdouble bonds to which nucleophilic groups, such as thiols, can be added,to form, for example, thioether bonds. These groups include maleimides,vinyl sulfones, vinyl pyridine, acrylates, methacrylates, andacrylamides. Other groups comprise leaving groups that can be displacedby a nucleophile; these include chloroethyl sulfone, pyridyl disulfides(which include a cleavable S—S bond), iodoacetamide, mesylate, tosylate,thiosulfonate, and tresylate. Epoxides react by ring opening by anucleophile, to form, for example, an ether or amine bond. Reactionsinvolving complementary reactive groups such as those noted above on theoligomer and the small molecule are utilized to prepare the conjugatesof the invention.

In some instances the heteropentacyclic nucleoside may not have afunctional group suited for conjugation. In this instance, it ispossible to modify (or “functionalize”) the “original” heteropentacyclicnucleoside so that it does have a functional group suited forconjugation. For example, if the heteropentacyclic nucleoside has anamide group, but an amine group is desired; it is possible to modify theamide group to an amine group by way of a Hofmann rearrangement, Curtiusrearrangement (once the amide is converted to an azide) or Lossenrearrangement (once amide is concerted to hydroxamide followed bytreatment with tolyene-2-sulfonyl chloride/base).

It is possible to prepare a conjugate of a heteropentacyclic nucleosidebearing a carboxyl group wherein the carboxyl group-bearing smallmolecule heteropentacyclic nucleoside is coupled to an amino-terminatedoligomeric ethylene glycol, to provide a conjugate having an amide groupcovalently linking the heteropentacyclic nucleoside to the oligomer.This can be performed, for example, by combining the carboxylgroup-bearing small molecule agent with the amino-terminated oligomericethylene glycol in the presence of a coupling reagent, (such asdicyclohexylcarbodiimide or “DCC”) in an anhydrous organic solvent.

Further, it is possible to prepare a conjugate of a heteropentacyclicnucleoside bearing a hydroxyl group wherein the hydroxyl group-bearingheteropentacyclic nucleoside is coupled to an oligomeric ethylene glycolhalide to result in an ether (—O—) linked small molecule conjugate. Thiscan be performed, for example, by using sodium hydride to deprotonatethe hydroxyl group followed by reaction with a halide-terminatedoligomeric ethylene glycol.

In another example, it is possible to prepare a conjugate of aheteropentacyclic nucleoside bearing a ketone group by first reducingthe ketone group to form the corresponding hydroxyl group. Thereafter,the heteropentacyclic nucleoside now bearing a hydroxyl group can becoupled as described herein.

In still another instance, it is possible to prepare a conjugate of aheteropentacyclic nucleoside bearing an amine group. In one approach,the amine group-bearing small molecule compound and an aldehyde-bearingoligomer are dissolved in a suitable buffer after which a suitablereducing agent (e.g., NaCNBH₃) is added. Following reduction, the resultis an amine linkage formed between the amine group of the aminegroup-containing small molecule of interest and the carbonyl carbon ofthe aldehyde-bearing oligomer.

In another approach for preparing a conjugate of a heteropentacyclicnucleoside bearing an amine group, a carboxylic acid-bearing oligomerand the amine group-bearing small molecule of interest are combined,typically in the presence of a coupling reagent (e.g., DCC). The resultis an amide linkage formed between the amine group of the aminegroup-containing small molecule and the carbonyl of interest thecarboxylic acid-bearing oligomer.

Exemplary compounds of the invention include those having the followingstructure:

wherein:

(HPC) is a five-membered heterocyclic moiety, preferably unsaturated andpreferably containing from one to three nitrogen atoms, more preferablyselected from the group consisting of

wherein R¹ is —C≡N, —C(O)NH₂, —C(S)NH₂, —C(O)OCH₃, —C(NH)NH₂ and—N(NH)N(H)OH;

R² is hydrogen or hydroxyl;

R³ is hydroxyl when R² is hydrogen and R³ is hydrogen when R² ishydroxyl;

X is a spacer moiety; and

POLY is a water-soluble, non-peptidic oligomer,

and 5′-phosphates and 3′,5′-cyclic phosphates thereof and ammonium andalkali metal salts of each of the 5′-phosphates and 3′,5′-cyclicphosphates.

Additional exemplary compounds of the invention include those having thefollowing structure:

wherein:

(HPC)-X-[POLY]_(a) is a five-membered heterocyclic moiety, preferablyunsaturated and preferably containing from one to three nitrogen atomsto which attached is one (when a=1) or two (when a=2) POLY species (eachPOLY being a water-soluble, non-peptidic oligomer), through X, a spacermoiety, R² is hydrogen or hydroxyl; and R³ is hydroxyl when R² ishydrogen and R³ is hydrogen when R² is hydroxyl; and 5′-phosphates and3′,5′-cyclic phosphates thereof and ammonium and alkali metal salts ofeach of the 5′-phosphates and 3′,5′-cyclic phosphates. Preferably, priorto any attachment of a water-soluble, non-peptidic oligomer through thespacer moiety, the five-membered ring is preferably selected from thegroup consisting of

wherein R¹ is —C≡N, —C(O)NH₂, —C(S)NH₂, —C(O)OCH₃, —C(NH)NH₂ and—N(NH)N(H)OH.

Further exemplary compounds of the invention include those having thefollowing structure:

wherein:

(HPC)-X-POLY is a five-membered heterocyclic moiety, preferablyunsaturated and preferably containing from one to three nitrogen atomsto which is attached a POLY, water-soluble, non-peptidic oligomer,through X, a spacer moiety, R² is hydrogen or hydroxyl; and R³ ishydroxyl when R² is hydrogen and R³ is hydrogen when R² is hydroxyl; and5′-phosphates and 3′,5′-cyclic phosphates thereof and ammonium andalkali metal salts of each of the 5′-phosphates and 3′,5′-cyclicphosphates. Preferably, prior to any attachment of a water-soluble,non-peptidic oligomer through the spacer moiety, the five-membered ringis preferably selected from the group consisting of

wherein R¹ is —C≡N, —C(O)NH₂, —C(S)NH₂, —C(O)OCH₃, —C(NH)NH₂ and—N(NH)N(H)OH.

Further exemplary compounds of the invention include those having thefollowing structure:

wherein:

R² is hydrogen or hydroxyl;

R³ is hydroxyl when R² is hydrogen and R³ is hydrogen when R² ishydroxyl;

X is a spacer moiety;

each POLY is independently a water-soluble, non-peptidic oligomer;

and (a) is one or two,

and 5′-phosphates and 3′,5′-cyclic phosphates thereof and ammonium andalkali metal salts of each of the 5′-phosphates and 3′,5′-cyclicphosphates.

Further additional exemplary compounds of the invention include thosehaving the following structure:

wherein:

R² is hydrogen or hydroxyl;

R³ is hydroxyl when R² is hydrogen and R³ is hydrogen when R² ishydroxyl;

X is a spacer moiety; and

POLY is a water-soluble, non-peptidic oligomer,

and 5′-phosphates and 3′,5′-cyclic phosphates thereof and ammonium andalkali metal salts of each of the 5′-phosphates and 3′,5′-cyclicphosphates.

Further exemplary compounds of the invention include those having thefollowing structure:

wherein:

R² is hydrogen or hydroxyl;

R³ is hydroxyl when R² is hydrogen and R³ is hydrogen when R² ishydroxyl;

each X is independently a spacer moiety;

each POLY is independently a water-soluble, non-peptidic oligomer;

and (a) is one or two,

and 5′-phosphates and 3′,5′-cyclic phosphates thereof and ammonium andalkali metal salts of each of the 5′-phosphates and 3′,5′-cyclicphosphates.

Further exemplary compounds of the invention include those having thefollowing structure:

wherein:

R² is hydrogen or hydroxyl;

R³ is hydroxyl when R² is hydrogen and R³ is hydrogen when R² ishydroxyl;

each X is independently a spacer moiety; and

each POLY is independently a water-soluble, non-peptidic oligomer,

and 5′-phosphates and 3′,5′-cyclic phosphates thereof and ammonium andalkali metal salts of each of the 5′-phosphates and 3′,5′-cyclicphosphates.

With respect to identifying the optimally sized oligomer for a givencompound, the following process can be followed.

First, an oligomer obtained from a monodisperse or bimodal water-solubleoligomer is conjugated to the small molecule drug. Preferably, the drugand the conjugate is orally bioavailable. Next, the metabolism of thedrug bearing a water-soluble oligomer is determined using an appropriatemodel and compared to that of the drug lacking the water-solubleoligomer. If the results are favorable, that is to say, if, for example,the rate of metabolism is reduced in the drug bearing the water-solubleoligomer, the above steps are repeated one or more times using oligomersof the same monomer type but having a different number of subunits andthe results compared. By making incremental changes in oligomer size andutilizing an experimental design approach, one can effectively identifya size of water-soluble oligomer that has an optimal reduction ofmetabolism when present in the drug bearing that size oligomer.

Animal models (rodents and dogs) can also be used to study oral drugtransport. In addition, non-in vivo methods include rodent everted gutexcised tissue and Caco-2 cell monolayer tissue-culture models. Thesemodels are useful in predicting oral drug bioavailability.

To determine whether the heteropentacyclic nucleoside itself or theconjugate of a heteropentacyclic nucleoside has activity, it is possibleto test such a compound. One having ordinary skill in the art using, forexample, the approach followed in Example 2 can determine such activity.

The present invention also includes pharmaceutical preparationscomprising a conjugate as provided herein in combination with apharmaceutical excipient. Generally, the conjugate itself will be in asolid form (e.g., a precipitate), which can be combined with a suitablepharmaceutical excipient that can be in either solid or liquid form.

Exemplary excipients include, without limitation, those selected fromthe group consisting of carbohydrates, inorganic salts, antimicrobialagents, antioxidants, surfactants, buffers, acids, bases, andcombinations thereof.

A carbohydrate such as a sugar, a derivatized sugar such as an alditol,aldonic acid, an esterified sugar, and/or a sugar polymer may be presentas an excipient. Specific carbohydrate excipients include, for example:monosaccharides, such as fructose, maltose, galactose, glucose,D-mannose, sorbose, and the like; disaccharides, such as lactose,sucrose, trehalose, cellobiose, and the like; polysaccharides, such asraffinose, melezitose, maltodextrins, dextrans, starches, and the like;and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol,sorbitol (glucitol), pyranosyl sorbitol, myoinositol, and the like.

The excipient can also include an inorganic salt or buffer such ascitric acid, sodium chloride, potassium chloride, sodium sulfate,potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic,and combinations thereof.

The preparation may also include an antimicrobial agent for preventingor deterring microbial growth. Nonlimiting examples of antimicrobialagents suitable for the present invention include benzalkonium chloride,benzethonium chloride, benzyl alcohol, cetylpyridinium chloride,chlorobutanol, phenol, phenylethyl alcohol, phenylmercuric nitrate,thimersol, and combinations thereof.

An antioxidant can be present in the preparation as well. Antioxidantsare used to prevent oxidation, thereby preventing the deterioration ofthe conjugate or other components of the preparation. Suitableantioxidants for use in the present invention include, for example,ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene,hypophosphorous acid, monothioglycerol, propyl gallate, sodiumbisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, andcombinations thereof.

A surfactant may be present as an excipient. Exemplary surfactantsinclude: polysorbates, such as “Tween 20” and “Tween 80,” and pluronicssuch as F68 and F88 (both of which are available from BASF, Mount Olive,N.J.); sorbitan esters; lipids, such as phospholipids such as lecithinand other phosphatidylcholines, phosphatidylethanolamines (althoughpreferably not in liposomal form), fatty acids and fatty esters;steroids, such as cholesterol; and chelating agents, such as EDTA, zincand other such suitable cations.

Pharmaceutically acceptable acids or bases may be present as anexcipient in the preparation. Nonlimiting examples of acids that can beused include those acids selected from the group consisting ofhydrochloric acid, acetic acid, phosphoric acid, citric acid, malicacid, lactic acid, formic acid, trichloroacetic acid, nitric acid,perchloric acid, phosphoric acid, sulfuric acid, fumaric acid, andcombinations thereof. Examples of suitable bases include, withoutlimitation, bases selected from the group consisting of sodiumhydroxide, sodium acetate, ammonium hydroxide, potassium hydroxide,ammonium acetate, potassium acetate, sodium phosphate, potassiumphosphate, sodium citrate, sodium formate, sodium sulfate, potassiumsulfate, potassium fumerate, and combinations thereof.

The amount of the conjugate in the composition will vary depending on anumber of factors, but will optimally be a therapeutically effectivedose when the composition is stored in a unit dose container. Atherapeutically effective dose can be determined experimentally byrepeated administration of increasing amounts of the conjugate in orderto determine which amount produces a clinically desired endpoint.

The amount of any individual excipient in the composition will varydepending on the activity of the excipient and particular needs of thecomposition. Typically, the optimal amount of any individual excipientis determined through routine experimentation, i.e., by preparingcompositions containing varying amounts of the excipient (ranging fromlow to high), examining the stability and other parameters, and thendetermining the range at which optimal performance is attained with nosignificant adverse effects.

Generally, however, excipients will be present in the composition in anamount of about 1% to about 99% by weight, preferably from about 5%-98%by weight, more preferably from about 15-95% by weight of the excipient,with concentrations less than 30% by weight most preferred.

These foregoing pharmaceutical excipients along with other excipientsand general teachings regarding pharmaceutical compositions aredescribed in “Remington: The Science & Practice of Pharmacy”, 19^(th)ed., Williams & Williams, (1995), the “Physician's Desk Reference”,52^(nd) ed., Medical Economics, Montvale, N.J. (1998), and Kibbe, A. H.,Handbook of Pharmaceutical Excipients, 3^(rd) Edition, AmericanPharmaceutical Association, Washington, D.C., 2000.

The pharmaceutical compositions can take any number of forms and theinvention is not limited in this regard. Exemplary preparations are mostpreferably in a form suitable for oral administration such as a tablet,caplet, capsule, gel cap, troche, dispersion, suspension, solution,elixir, syrup, lozenge, transdermal patch, spray, suppository, andpowder.

Oral dosage forms are preferred for those conjugates that are orallyactive, and include tablets, caplets, capsules, gel caps, suspensions,solutions, elixirs, and syrups, and can also comprise a plurality ofgranules, beads, powders or pellets that are optionally encapsulated.Such dosage forms are prepared using conventional methods known to thosein the field of pharmaceutical formulation and described in thepertinent texts.

Tablets and caplets, for example, can be manufactured using standardtablet processing procedures and equipment. Direct compression andgranulation techniques are preferred when preparing tablets or capletscontaining the conjugates described herein. In addition to theconjugate, the tablets and caplets will generally contain inactive,pharmaceutically acceptable carrier materials such as binders,lubricants, disintegrants, fillers, stabilizers, surfactants, coloringagents, flow agents, and the like. Binders are used to impart cohesivequalities to a tablet, and thus ensure that the tablet remains intact.Suitable binder materials include, but are not limited to, starch(including corn starch and pregelatinized starch), gelatin, sugars(including sucrose, glucose, dextrose and lactose), polyethylene glycol,waxes, and natural and synthetic gums, e.g., acacia sodium alginate,polyvinylpyrrolidone, cellulosic polymers (including hydroxypropylcellulose, hydroxypropyl methylcellulose, methyl cellulose,microcrystalline cellulose, ethyl cellulose, hydroxyethyl cellulose, andthe like), and Veegum. Lubricants are used to facilitate tabletmanufacture, promoting powder flow and preventing particle capping(i.e., particle breakage) when pressure is relieved. Useful lubricantsare magnesium stearate, calcium stearate, and stearic acid.Disintegrants are used to facilitate disintegration of the tablet, andare generally starches, clays, celluloses, algins, gums, or crosslinkedpolymers. Fillers include, for example, materials such as silicondioxide, titanium dioxide, alumina, talc, kaolin, powdered cellulose,and microcrystalline cellulose, as well as soluble materials such asmannitol, urea, sucrose, lactose, dextrose, sodium chloride, andsorbitol. Stabilizers, as well known in the art, are used to inhibit orretard drug decomposition reactions that include, by way of example,oxidative reactions.

Capsules are also preferred oral dosage forms, in which case theconjugate-containing composition can be encapsulated in the form of aliquid or gel (e.g., in the case of a gel cap) or solid (includingparticulates such as granules, beads, powders or pellets). Suitablecapsules include hard and soft capsules, and are generally made ofgelatin, starch, or a cellulosic material. Two-piece hard gelatincapsules are preferably sealed, such as with gelatin bands or the like.

Included are parenteral formulations in the substantially dry form(typically as a lyophilizate or precipitate, which can be in the form ofa powder or cake), as well as formulations prepared for injection, whichare typically liquid and requires the step of reconstituting the dryform of parenteral formulation. Examples of suitable diluents forreconstituting solid compositions prior to injection includebacteriostatic water for injection, dextrose 5% in water,phosphate-buffered saline, Ringer's solution, saline, sterile water,deionized water, and combinations thereof.

In some cases, compositions intended for parenteral administration cantake the form of nonaqueous solutions, suspensions, or emulsions, eachtypically being sterile. Examples of nonaqueous solvents or vehicles arepropylene glycol, polyethylene glycol, vegetable oils, such as olive oiland corn oil, gelatin, and injectable organic esters such as ethyloleate.

The parenteral formulations described herein can also contain adjuvantssuch as preserving, wetting, emulsifying, and dispersing agents. Theformulations are rendered sterile by incorporation of a sterilizingagent, filtration through a bacteria-retaining filter, irradiation, orheat.

The conjugate can also be administered through the skin usingconventional transdermal patch or other transdermal delivery system,wherein the conjugate is contained within a laminated structure thatserves as a drug delivery device to be affixed to the skin. In such astructure, the conjugate is contained in a layer, or “reservoir,”underlying an upper backing layer. The laminated structure can contain asingle reservoir, or it can contain multiple reservoirs.

The conjugate can also be formulated into a suppository for rectaladministration. With respect to suppositories, the conjugate is mixedwith a suppository base material which is (e.g., an excipient thatremains solid at room temperature but softens, melts or dissolves atbody temperature) such as coca butter (theobroma oil), polyethyleneglycols, glycerinated gelatin, fatty acids, and combinations thereof.Suppositories can be prepared by, for example, performing the followingsteps (not necessarily in the order presented): melting the suppositorybase material to form a melt; incorporating the conjugate (either beforeor after melting of the suppository base material); pouring the meltinto a mold; cooling the melt (e.g., placing the melt-containing mold ina room temperature environment) to thereby form suppositories; andremoving the suppositories from the mold.

The invention also provides a method for administering a conjugate asprovided herein to a patient suffering from a condition that isresponsive to treatment with the conjugate. The method comprisesadministering, generally orally, a therapeutically effective amount ofthe conjugate (preferably provided as part of a pharmaceuticalpreparation). Other modes of administration are also contemplated, suchas pulmonary, nasal, buccal, rectal, sublingual, transdermal, andparenteral. As used herein, the term “parenteral” includes subcutaneous,intravenous, intra-arterial, intraperitoneal, intracardiac, intrathecal,and intramuscular injection, as well as infusion injections.

In instances where parenteral administration is utilized, it may benecessary to employ somewhat bigger oligomers than those describedpreviously, with molecular weights ranging from about 500 to 30K Daltons(e.g., having molecular weights of about 500, 1000, 2000, 2500, 3000,5000, 7500, 10000, 15000, 20000, 25000, 30000 or even more).

The method of administering may be used to treat any condition that canbe remedied or prevented by administration of the particular conjugate.Those of ordinary skill in the art appreciate which conditions aspecific conjugate can effectively treat. The actual dose to beadministered will vary depend upon the age, weight, and generalcondition of the subject as well as the severity of the condition beingtreated, the judgment of the health care professional, and conjugatebeing administered. Therapeutically effective amounts are known to thoseskilled in the art and/or are described in the pertinent reference textsand literature. Generally, a therapeutically effective amount will rangefrom about 0.001 mg to 1000 mg, preferably in doses from 0.01 mg/day to750 mg/day, and more preferably in doses from 0.10 mg/day to 500 mg/day.

The unit dosage of any given conjugate (again, preferably provided aspart of a pharmaceutical preparation) can be administered in a varietyof dosing schedules depending on the judgment of the clinician, needs ofthe patient, and so forth. The specific dosing schedule will be known bythose of ordinary skill in the art or can be determined experimentallyusing routine methods. Exemplary dosing schedules include, withoutlimitation, administration five times a day, four times a day, threetimes a day, twice daily, once daily, three times weekly, twice weekly,once weekly, twice monthly, once monthly, and any combination thereof.Once the clinical endpoint has been achieved, dosing of the compositionis halted.

One advantage of administering the conjugates of the present inventionis that a reduction in first pass metabolism may be achieved relative tothe parent drug. Such a result is advantageous for many orallyadministered drugs that are substantially metabolized by passage throughthe gut. In this way, clearance of the conjugate can be modulated byselecting the oligomer molecular size, linkage, and position of covalentattachment providing the desired clearance properties. One of ordinaryskill in the art can determine the ideal molecular size of the oligomerbased upon the teachings herein. Preferred reductions in first passmetabolism for a conjugate as compared to the correspondingnonconjugated small drug molecule include: at least about 10%, at leastabout 20%, at least about 30; at least about 40; at least about 50%; atleast about 60%, at least about 70%, at least about 80% and at leastabout 90%.

Thus, the invention provides a method for reducing the metabolism of anactive agent. The method comprises the steps of: providing monodisperseor bimodal conjugates, each conjugate comprised of a moiety derived froma small molecule drug covalently attached by a stable linkage to awater-soluble oligomer, wherein said conjugate exhibits a reduced rateof metabolism as compared to the rate of metabolism of the smallmolecule drug not attached to the water-soluble oligomer; andadministering the conjugate to a patient. Typically, administration iscarried out via one type of administration selected from the groupconsisting of oral administration, transdermal administration, buccaladministration, transmucosal administration, vaginal administration,rectal administration, parenteral administration, and pulmonaryadministration.

Although useful in reducing many types of metabolism (including bothPhase I and Phase II metabolism) can be reduced, the conjugates areparticularly useful when the small molecule drug is metabolized by ahepatic enzyme (e.g., one or more of the cytochrome P450 isoforms)and/or by one or more intestinal enzymes.

All articles, books, patents, patent publications and other publicationsreferenced herein are incorporated by reference in their entireties. Inthe event of an inconsistency between the teachings of thisspecification and the art incorporated by reference, the meaning of theteachings in this specification shall prevail.

EXPERIMENTAL

It is to be understood that while the invention has been described inconjunction with certain preferred and specific embodiments, theforegoing description as well as the examples that follow are intendedto illustrate and not limit the scope of the invention. Other aspects,advantages and modifications within the scope of the invention will beapparent to those skilled in the art to which the invention pertains.

All chemical reagents referred to in the appended examples arecommercially available unless otherwise indicated. The preparation ofPEG-mers is described in, for example, U.S. Patent ApplicationPublication No. 2005/0136031.

All ¹H NMR (nuclear magnetic resonance) data was generated by NMRspectrometer manufactured by Bruker. A list of certain compounds as wellas the source of the compounds is provided below.

EXAMPLE 1 Exemplary PEG-Heteropentacyclic Nucleoside Conjugates

The structures of exemplary compounds of the present invention areprovided below:

wherein each appearance of (n) is independently a variable from betweenabout 1 and about 30 (inclusive).

For the compounds prepared in this example, the following materials wereused. Ribavirin was obtained from Yuhan Corporation, plant 678-1,Sungkok-Dong, Ansan-Shi Kyunggi-Do, Korea. 2,2-Dimethoxypropane wasobtained from Aldrich, 98%, Lot No. 06808PD (St. Louis, Mo.).P-Toluenesulfonic acid monohydrate was obtained from Aldrich, 98.5%, LotNo. 07204EC (St. Louis, Mo.). tert-Butyldimethylsilyl chloride wasobtained from Aldrich, 97%, Batch #: 12009DC (St. Louis, Mo.).tert-butylchlorodiphenylsilane was obtained from Aldrich, 98%, Batch #:21017HB (St. Louis, Mo.). Imidazole was obtained from Aldrich, 99%,Batch #: 11721TD (St. Louis, Mo.). Pyridine was obtained from Aldrich,Batch #: 03859ED (St. Louis, Mo.). Phosphorus oxychloride was obtainedfrom Aldrich, 99%, Batch #: 16817BD (St. Louis, Mo.). Triethylamine wasobtained from Aldrich, 99.5%, Batch #: 04623HD (St. Louis, Mo.). Sodiummethoxide (25 wt % solution in methanol) was obtained from Aldrich, LotNo. 08730JR (St. Louis, Mo.).1,3-Dichloro-1,1,3,3-tetraisopropyldisiloxane was obtained from Aldrich,97%, Lot No. 0615JD (St. Louis, Mo.). Dimethylaminopyridine was obtainedfrom Fluka, 98%. Sodium hydride (60% dispersion in mineral oil) wasobtained from Aldrich, Batch #10429PD (St. Louis, Mo.).

EXAMPLES 1A AND 1B Preparation of mPEG_(n)-O-2′-ribavirin anddi-mPEG_(n)-N, O-2′-ribavirin

An exemplary synthetic scheme to prepare mPEG_(n)-O-2′-ribavirin anddi-mPEGn-N, O-2′-ribavirin is schematically shown below.

Synthesis of 3,5-O-(1,1,3,3-tetraisopropyl-1,3-disiloxanyl)-ribavirin 2

Ribavirin 1 (203.4 mg, 0.83 mmol) was mixed with pyridine (3.0 mL) atroom temperature, and then 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane(0.33 mL, 1.00 mmol) was added. The resulting mixture was stirred atroom temperature for 23.5 hours. Water was added to quench the reactionand extracted with EtOAc (3×20 mL). The combined organic solution waswashed with brine (50 mL), dried over sodium sulfate, concentrated. Theresidue was purified by flash column chromatography on silica gel usingMeOH/DCM (0-5%) to afford the product (369 mg, 91%). ¹H-NMR (CDCl₃)δ8.36 (s, 1H, C₅H), 6.90 (bs, 1H, NH₂), 5.90 (s, 1H, C₁, H), 5.58 (bs,1H, NH₂), 4.66-4.61 (m, 1H), 4.45 (d, 1H), 4.17-3.97 (m, 3H), 2.93 (s,1H), 1.00-0.98 (m, 28H). LC-Ms: 487.3 MH⁺.

Synthesis of Compound 18 and Compound 19

NaH (in a dispersion in mineral oil) is added to a stirred solution ofcompound 2 in anhydrous THF at room temperature. Thereafter, mPEG_(n)-LV[wherein LV is a leaving group, such as a halogen (e.g., bromo andchloro) or sulfonate ester (e.g., mesylate) and n is an integer from 1to 30] is added. A mixture is formed and is subsequently stirred at roomtemperature for one day. Thereafter, the solvent is removed under highvacuum. The resulting residue is mixed with a mixture of EtOAc/water.The organic phase is then separated and the aqueous phase is extractedwith EtOAc (2×20 mL). The combined organic solutions are then washedwith brine (50 mL), dried over sodium sulfate, concentrated to afford aresidue. The residue is separated by using conventional techniques toresult in compound 18 and compound 19.

Synthesis of mPEG_(n)-O-2′-Ribavirin

Compound 18 is added to THF to which tetra-n-butylammonium fluoride(“TBAF”) is added. The resulting mixture is stirred at room temperature.The solvent is removed and mPEG_(n)-O-2′-Ribavirin is recovered usingconventional techniques.

Synthesis of Di-mPEG_(n)-N, O-2′-Ribavirin

Compound 19 is added to THF to which tetra-n-butylammonium fluoride(“TBAF”) is added. The resulting mixture is stirred at room temperature.The solvent is removed and Di-mPEG_(n)-N, O-2′-Ribavirin is recoveredusing conventional techniques.

EXAMPLE 1C Preparation of mPEG_(n)-N-ribavirin

An exemplary synthetic scheme to prepare mPEG_(n)-N-Ribavirin isschematically shown below.

wherein (n) is a variable from between about 1 and about 30 (inclusive).

Synthesis of1-(2′,3′-Di-O-isopropylidene-β-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide5

A suspension of ribavirin 1 (318 mg, 1.302 mmol) and p-toluenesulfonicacid monohydrate (“p-TsOH”) (41 mg, 0.212 mmol) was cooled to 0° C. inan ice-water bath. 2,2-Dimethoxypropane (0.18 mL, 1.438 mmol) was added.The suspension was stirred at 0° C. for 30 minutes, and then at roomtemperature for three hours. The mixture was still suspension. Then moreof acetone (5 mL) and 2,2-dimethoxypropane (0.4 mL) was added. Themixture was stirred at room temperature for 21 hours, and then heated at60° C. for one hour. The solution became clear. When the mixture wascooled to room temperature, 5% aqueous sodium bicarbonate (about 1 mL)was added to quench the reaction. Some precipitate was observed. Themixture was filtered and washed with acetone. The precipitate was driedunder high vacuum and checked by ¹H-NMR in DMSO. No peaks of interestwere observed. The solution was concentrated under reduced and theresidue was purified by column chromatography on SiO₂ using acetone/DCM(15%) and MeOH/acetone/DCM (2/15/83) to afford white solid as theproduct (305 mg, 82%).

Run 2, a suspension of ribavirin (1.05 g, 4.3 mmol), p-toluenesulfonicacid monohydrate (“p-TsOH”) (138 mg, 0.71 mmol) and 2,2-dimethoxypropane(1.0 mL, 7.99 mmol) in acetone (15 mL) was heated at 60° C. for threehours. The mixture was cooled to room temperature, and then 5% aqueoussodium bicarbonate (1 mL) was added to quench the reaction mixture. Theprecipitate was removed and the filtrate was collected and concentrated.The residue was purified by flash column chromatography on silica usingMeOH/DCM (0-10%) to afford the product (1.21 g, 99% yield) as a whitesolid.

Run 3, a suspension of ribavirin (4.15 g, 17.01 mmol), p-toluenesulfonicacid monohydrate (“p-TsOH”) (535 mg, 2.77 mmol) and 2,2-dimethoxypropane(3.4 mL, 27.16 mmol) in acetone (50 mL) was heated at 60° C. for fourhours. The mixture was cooled to room temperature, and then 5% aqueoussodium bicarbonate (2 mL) was added to quench the reaction mixture. Themixture was concentrated and the residue was dried under high vacuum.The residue was purified with biotage SP™ flash purification system onsilica using MeOH/DCM (0-10%) to afford the product (3.91 g, 81% yield)as a white solid.

¹H-NMR (DMSO-d₆), δ8.78 (s, 1H)), triazole ring proton), 7.84, 7.64 (2s,2H, CONH₂), 6.20 (d, J=1.5 Hz, 1H), C₁H), 5.18 (dd, J=1.5 Hz, J=6.0 Hz,1H), C₂.H), 4.90 (dd, J=1.8 Hz, J=6.0 Hz, 1H), C₃.H), 4.23 (dt, J=1.8Hz, J=6.0 Hz, 1H), C₄.H), 3.51-3.35 (m, 2H, C₅.H), 1.50, 1.32 (2s, 6H,isopropylidene methyls). LC-Ms, 285.1 (MH⁺).

Synthesis of1-[(5′-O-(4,4′-Dimethoxytrityl)-2′,3′-di-O-isopropylidene-β-D-ribofuranosyl]-1,2,4-triazole-3-carboxamide6

4,4′-Dimethoxytrityl chloride (156 mg, 0.44 mmol) was added to a stirredsolution of1-(2′,3′-di-O-isopropylidene-β-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide(compound 5) (111 mg, 0.39 mmol) in pyridine (3 mL). The resultingmixture was stirred at room temperature for 22 hours. More of4,4′-dimethoxytrityl chloride (71 mg) was added. After 7.5 hours at roomtemperature, DMAP (56 mg) was added. The mixture was stirred at roomtemperature for another 21 hours. MeOH (2 mL) was added to quench thereaction, diluted with water, extracted with EtOAc (3×20 mL). Theorganic solution was washed with brine, dried over Na₂SO₄, concentrated.The residue was purified by flash column chromatography on silica gelusing EtOAc/hexane (0-100%) to afford the product compound 6 (172 mg,75% yield). ¹H-NMR (CDCl₃), 88.20 (s, 1H), C₅H, triazole proton),7.30-7.11 (m, 9H, Ar—H), 6.76-6.72 (m, 4H, Ar—H), 6.86, 6.50 (2 bs, 2H,NH₂), 6.00 (d, J=0.9 Hz, 1H, C₁.H), 5.33 (dd, J=0.9-1.2 Hz, J=6.0 Hz,1H), 4.78 (dd, J=2.1 Hz, J=6.0 Hz, 1H), 4.54 (dt, J=1.5-1.8 Hz, J=5.7Hz, 1H), 3.72 (s, 6H, 2 CH₃), 3.21-3.12 (m, 2H), 1.55, 1.32 (2s, 6H, 2CH₃).

Synthesis of1-[5′-O-(4,4′-Dimethoxytrityl)-2′,3′-di-O-isopropylidene-β-D-ribofuranosyl]-N-mPEG₃-1,2,4-triazole-3-carboxamide7 (n=3)

NaH (60% dispersion in mineral oil, 50 mg, 1.25 mmol) was added to astirred solution of1-[5′-O-(4,4′-dimethoxytrityl)-2′,3′-di-O-isopropylidene-β-D-ribofuranosyl]-1,2,4-triazole-3-carboxamide(compound 6) (153 mg, 0.26 mmol) in anhydrous THF (2 mL) at roomtemperature. Thereafter, mPEG₃-Br (135 mg, 0.59 mmol) was added. Theresulting mixture was stirred at room temperature for 24.5 hours. Thesolvent was removed under high vacuum. The residue was mixed with amixture of EtOAc/water (40 mL, 50%). The organic phase was separated andthe aqueous phase was extracted with EtOAc (2×20 mL). The combinedorganic solution was washed with brine (50 mL), dried over sodiumsulfate, concentrated to afford a residue. The residue was separated bypreparative TLC using MeOH/EtOAc (10%) to result in the product (67 mg),as well as a mixture of the product and an impurity (31 mg). ¹H-NMR(CDCl₃) for compound 7 (n=3): δ8.22 (s, 1H, C₅H, triazole proton), 7.37(br, 1H, NH), 7.30-7.13 (m, 9H, Ar—H), 6.77-6.74 (m, 4H, Ar—H), 5.99 (s,1H), 5.35 (d, J=6.0 Hz, 1H), 4.74 (dd, J=2.1 Hz, J=6.0 Hz, 1H), 4.52 (m,1H), 3.76 (s, 6H, 2 CH₃), 3.64-3.61 (m, 10H), 3.53-3.47 (m, 2H), 3.30(s, 3H, CH₃), 3.13-3.10 (m, 2H, CH₂), 1.55, 1.32 (2s, 6H, 2CH₃). LC-MS:755.4 (MNa⁺).

Synthesis of mPEG₃-N-Ribavirin

1.0 M HCl in ether (10 mL) was added to a stirred solution of compound7) (n=3) in methanol (6 mL) at room temperature. The resulting mixturewas stirred at room temperature for several hours. The mixture was thenconcentrated to remove the solvent and HCl. The residue was purified byprep TLC (10% MeOH/DCM) to afford mPEG₃-N-Ribavirin. NMR data notavailable.

Synthesis of mPEG₅-N-Ribavirin

NaH (60% dispersion in mineral oil, 1.273 g, 4.039 mmol) was added to astirred solution of1-[5′-O-(4,4′-dimethoxytrityl)-2′,3′-di-O-isopropylidene-β-D-ribofuranosyl]-1,2,4-triazole-3-carboxamide(compound 6) (778 mg, 1.328 mmol) in anhydrous THF (6 mL) at roomtemperature. Thereafter, mPEG₅-Br (1.273 g, 4.039 mmol) was added. Theresulting mixture was stirred at room temperature for 27.5 hours.Saturated NH₄Cl (˜3 mL) was added to quench the reaction. The solventswere removed under high vacuum. The residue was mixed with a mixture ofaq. 5% NaHCO₃ solution (40 mL) and DCM (25 mL). The organic phase wasseparated and the aqueous phase was extracted with DCM (3×25 mL). Thecombined organic solution was washed with brine, dried over sodiumsulfate, concentrated to afford 1.8207 g of crude product.

The crude product was treated with 80% HOAc solution for 23 hours, at45° C. for 4.5 hours, and then concentrated to remove the solvents.Purification of the crude residue with flash column chromatography onsilica gel resulted in mPEG₅-N-ribavirin (n=5) (129 mg) (“first lot”)and an impure product (503 mg). 40 mL of 1.0 m HCl in ether solution wasadded to stirred solution of the above impure product in methanol (10mL) at 0° C. The resulting mixture was stirred at 0° C. for 80 minutes.The solvents and hydrochloride were evaporated under reduced pressure toafford a residue. The residue was purified with reverse phase columnchromatography using acetonitrile in water to afford mPEG₅-N-ribavirin(90.4 mg) (“second lot”) and di-mPEG₅-N-ribavirin (compound 17) (128 mg)(See “Example 1D”).

¹H-NMR (CDCl₃) for mPEG₅-N-ribavirin (n=5) (“first lot”): 8.51 (s, 1H,C₅H, triazole proton), 7.72 (br, 1H, NH), 5.90 (d, J=3.3 Hz, 1H), 4.75(br, 1H), 4.63 (m, 1H), 4.50 (m, 1H), 4.22 (m, 1H), 4.15 (br, 1H),3.95-3.76 (m, 3H), 3.63-3.51 (m, 20H), 3.35 (s, 3H, CH₃). LC-MS: 479.3(MH⁺).

¹H-NMR (CDCl₃) for mPEG₅-N-ribavirin 17 (n=5) (“second lot”): 8.36 (s,1H, C₅H, triazole proton), 5.89 (d, J=2.4 Hz, 1H), 4.57 (m, 2H),4.35-4.08 (m, 3H), 3.87 (m, 3H), 337-3.52 (m, 40H), 3.363 (s, 3H, CH₃),3.357 (s, 3H, CH₃). LC-MS: 713.5 (MH⁺).

EXAMPLE 1D Synthesis of Di-mPEG_(n)-N-ribavirin

An exemplary synthetic scheme to prepare Di-mPEG_(n)-N-Ribavirin isschematically shown below.

wherein (n) is a variable from between about 1 and about 30 (inclusive).

Synthesis of1-[5′-O-(4,4′-Dimethoxytrityl)-2′,3′-di-O-isopropylidene-β-D-ribofuranosyl]-N-di-mPEG₃-1,2,4-triazole-3-carboxamide14 (n=3)

NaH (60% dispersion in mineral oil, 110 mg, 2.75 mmol) was added to astirred solution of1-[5′-O-(4,4′-dimethoxytrityl)-2′,3′-di-O-isopropylidene-β-D-ribofuranosyl]-1,2,4-triazole-3-carboxamide(compound 6, made in accordance in Example IC) (438 mg, 0.747 mmol) inanhydrous THF (5 mL) at room temperature. Then, mPEG₃-Br (510 mg, 0.246mmol) was added. The resulting mixture was stirred at room temperaturefor 23 hours. Sat NH₄Cl mL) was added to quench the reaction. Thesolvent was removed under high vacuum. The residue was mixed with amixture of EtOAc/water (50 mL, 50%). The organic phase was separated andthe aqueous phase was extracted with EtOAc (2×25 mL). The combinedorganic solution was washed with brine, dried over sodium sulfate,concentrated to afford a residue. The residue was separated by flashcolumn chromatography on silica gel using EtOAc/hexane (50-100%) andMeOH/EtOAc (5%, 10%) or MeOH/DCM (0-10%) to result in mono-PEG compound7 (n=3) (92 mg), di-PEG compound 14 (n=3) (227 mg), de-DMTr groupcompound 15 (n=3) (33.4 mg) and compound 17 (53 mg). ¹H-NMR (CDCl₃) forcompound 14: δ8.22 (s, 1H, C₅H, triazole proton), 7.34-7.16 (m, 9H,Ar—H), 6.79-6.75 (m, 4H, Ar—H), 5.98 (s, 1H), 5.19 (dd, J=1.5 Hz, J=6.0Hz, 1H), 4.73 (ds, J=2.7 Hz, J=6.0 Hz, 1H), 4.46 (m, 1H), 3.76 (s, 6H, 2CH₃), 3.75-3.46 (m, 24H), 3.35 (s, 3H, CH₃), 3.34 (s, 3H, CH₃),3.22-3.13 (m, 2H, CH₂), 1.55, 1.32 (2s, 6H, 2 CH₃). LC-MS: 901.4 (MNa⁺).

Synthesis of Di-mPEG₃-N-ribavirin (17) (n=3)

Following the approach described with respect to the HCl acid treatmentof compound 14 to prepare Di-mPEG₃-N-ribavirin, compound 17 (n=3) wasafforded. No NMR data available.

Synthesis of Di-mPEG₅-N-ribavirin (17) (n=5)

Di-mPEG₅-N-ribavirin (17) (n=5) was prepared as described in Example 1C.

EXAMPLE 1D Preparation of mPEG_(n)-N-ribamidine

An exemplary synthetic scheme to prepare mPEG-N-Ribamidine isschematically shown below.

wherein (n) is a variable from between about 1 and about 30 (inclusive).

Synthesis of2′,3′,5′-O-triacetyl-β-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide 8

A suspension of ribavirin 1 (568 mg, 2.33 mmol) in acetic anhydride (4mL) and pyridine (1 mL) was stirred at room temperature for 22 hours.The mixture was concentrated under reduced pressure and the residue wasdried under high vacuum to afford the product in quantitative yield.

Run 2, a suspension of ribavirin (2.05 g, 8.39 mmol) in acetic anhydride(12 mL) and pyridine (4 mL) was stirred at room temperature for 18hours. The mixture was concentrated under reduced pressure and theresidue was dried under high vacuum to afford the product inquantitative yield.

¹H-NMR (DMSO-d₆), δ8.84 (s, 1H, C₅H, triazole proton), 7.90, 7.71 (2s,2H, CONH₂), 6.32 (d, J=3 Hz, 1H, C₁.H), 5.66 (m, 1H), 5.57 (t, J=5.4 Hz,1H), 4.42-4.37 (m, 2H), 4.10 (m, 1H), 2.10, 2.09, 2.01 (3 s, 9H, COCH₃).LC-Ms, 371.2 (MH⁺).

Synthesis of3-cyano-2′,3′,5′-O-triacetyl-β-D-ribofuranosyl)-1,2,4-triazole 9

A mixture of crude2,3′,5′-O-triacetyl-β-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide(compound 8) (934 mg) and triethylamine (5.5 mL) in chloroform (10 mL)was cooled to 0° C. in an ice-water bath. Phosporus oxychloride (0.7 mL,7.57 mmol) was added dropwise with stirring and the solution was allowedto warm to room temperature. After the mixture was stirred at roomtemperature for 17 hours, the brown reaction mixture was concentrated todryness in vacuum and the residue was dissolved in dichloromethane (20mL). The organic solution was washed with saturated aqueous sodiumbicarbonate (3×60 mL), dried over sodium sulfate, and concentrated. Theresidue was purified by column chromatography on silica gel usingMeOH/DCM (2%) to afford the product (0.718 g, 87% yield based onribavirin).

Run 2, a mixture of crude2′,3′,5′-O-triacetyl-β-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide(from 2.05 g of ribavirin) and triethylamine (20 mL) in chloroform (35mL) was cooled to 0° C. in an ice-water bath. Phosporus oxychloride (2.5mL, 27.04 mmol) was added dropwise with stirring. The solution wasstirred at 0° C. for one hour, the ice-water bath was removed and thesolution was stirred at room temperature for 17 hours. The brownreaction mixture was concentrated to dryness in vacuum. The residue waspurified by column chromatography on silica gel using MeOH/DCM (2%) toafford the product (2.533 g, 86% yield based on ribavirin).

¹H-NMR (CDCl₃), δ8.36 (s, 1H, C₅H, triazole proton), 5.99 (d, J=3.3 Hz,1H, C₁.H), 5.68 (m, 1H), 5.52 (t, J=5.4 Hz, 1H), 4.51-4.41 (m, 2H),4.24-4.18 (m, 1H), 2.13, 2.12, 2.09 (3 s, 9H, COCH₃). LC-Ms, 259.1[M-C₃HN₄]⁺, 375.2 (MNa⁺).

Synthesis of Methyl 1-β-D-Ribofuranosyl-1,2,4-triazole-3-carboximidate10

Sodium methoxide (25 wt % solution in methanol, 0.83 mL, 3.63 mmol) wasadded to a stirred solution of3-cyano-2′,3′,5′-O-triacetyl-β-D-ribofuranosyl)-1,2,4-triazole (compound9) (787 mg, 2.23 mmol) im methanol (15 mL). The mixture was stirred atroom temperature for 20 hours. Acetic acid (0.21 mL, 3.70 mmol) wasadded to quench the reaction. The mixture was concentrated under reducedpressure to afford the crude product. The crude was directly used forthe next reaction without purification. ¹H-NMR (DMSO-d₆), δ 8.90 (s, 1H,C₅H, triazole proton), 8.82 (bs, 1H, NH), 5.81 (d, J=3.9 Hz, 1H, C₁.H),4.34 (t, J=4.1-4.5 Hz, 1H), 4.13 (t, J=4.8-5.1 Hz, 1H), 3.93 (q,J=4.5-4.8 Hz, 1H), 3.83 (s. 3H, OCH₃), 3.64-3.43 (m, 2H) and other sugarprotons. LC-Ms, 259.1 (MH⁺).

Synthesis of mPEG_(n)-ribamidine 11 (n=3)

mPEG₃-NH₂ (147 mg, 0.90 mmol) was added to a stirred solution of crudemethyl amidate 10 (290 mg, ˜0.56 mmol)) in methanol (5.0 mL) at roomtemperature. The resulting mixture was heated at 45° C. for 15 hours.The mixture was cooled to room temperature and concentrated underreduced pressure. The residue was triturated with EtOAc, diluted withethyl ether, centrifuged. The precipitate was triturated again with DCM,diluted with ethyl ether and centrifuged. The precipitate was collectedand dried under high vacuum to afford 156 mg of product. ¹H-NMR (MeOD)for compound 11 (n=3): 8.81 (s, 1H, C₅H, triazole proton), 5.92 (d,J=3.6 Hz, 1H), 4.487 (m, 1H), 4.333 (m, 1H), 4.132 (m, 1H), 3.87-3.53(m, 19H), 3.358 (s, 3H, CH₃). LC-MS: 390.2 (MH⁺).

Synthesis of mPEG_(n)-ribamidine 11 (n=6)

mPEG₆-NH₂ (330 mg, 1.12 mmol) was added to a stirred solution of crudemethyl amidate 10 (250 mg, ˜0.97 mmol)) in methanol (6.0 mL) at roomtemperature. The resulting mixture was heated at 45° C. for 17 hours.The mixture was cooled to room temperature and concentrated underreduced pressure. The residue was triturated with EtOAc, diluted withethyl ether, centrifuged. The precipitate was triturated again with DCM,diluted with ethyl ether and centrifuged. The precipitate was collectedand dried under high vacuum to afford 126 mg of product. ¹H-NMR (MeOD)for compound 11 (n=6): 8.59 (s, 1H, C₅H, triazole proton), 5.71 (d,J=3.6 Hz, 1H), 4.29 (m, 1H), 4.14 (m, 1H), 3.91 (m, 1H), 3.65-3.31 (m,31H), 3.15 (s, 3H, CH₃). LC-MS: 522.3 (MH⁺).

Synthesis of mPEG_(n)-ribamidine 11 (n=7)

mPEG₇-NH₂ (565 mg, 1.66 mmol) was added to a stirred solution of crudemethyl amidate 10 (346 mg, ˜1.34 mmol)) in methanol (6.0 mL) at roomtemperature. The resulting mixture was heated at 45° C. for 15.5 hours.The mixture was concentrated under reduced pressure. The residue wastriturated with EtOAc, diluted with ethyl ether, centrifuged. Theprecipitate was triturated again with DCM, diluted with ethyl ether andcentrifuged. The precipitate was collected and dried under high vacuumto afford 431 mg of product. ¹H-NMR (MeOD) for compound 11: 8.61 (s, 1H,C₅H, triazole proton), 5.71 (d, J=3.3 Hz, 1H), 4.29 (m, 1H), 4.14 (m,1H), 3.91 (m, 1H), 3.75-3.33 (m, 35H), 3.15 (s, 3H, CH₃). LC-MS: 566.3(MH⁺).

EXAMPLE 2 Antiviral Activity

Ribavirin is a guanosine analogue with broad antiviral activity. Theantiviral activities of various compounds prepared in accordance withExample 1 were tested in two in vitro models of viral infection.

Several compounds prepared in accordance with Example 1 were solubilizedin DMSO at 40 mM. The compounds were evaluated using a 100 μM high testconcentration and serially diluted in half-log increments for the invitro antiviral assay. Ribavirin (anti-influenza and anti-BVDV) andamantadine (anti-influenza) were purchased from Sigma (St. Louis, Mo.)and used as a positive control compound in the cytoprotection assays.

Anti-Influenza Assay:

Cell Preparation—MDCK cells (female cocker spaniel kidney epithelial;ATCC catalog no. CCL-34) were passaged in T-75 flasks prior to use inthe antiviral assay. On the day preceding the assay, the cells weresplit 1:2 to assure they were in an exponential growth phase at the timeof infection. Total cell and viability quantification was performedusing a hemocytometer and Trypan Blue dye exclusion. Cell viability wasgreater than 95% for the cells to be utilized in the assay. The cellswere resuspended at 1×10⁵ cells/mL in tissue culture medium (Dulbecco'sModified Eagles Medium, “DMEM,” supplemented with 10% heat inactivatedfetal bovine serum, 2 mmol/L L-glutamine, 100 U/mL penicillin, 100 μg/mLstreptomycin, 0.1 mM NEAA, and 1 mM sodium pyruvate) to thedrug-containing microtiter plates in a volume of 100 μL. The microtiterplates were incubated at 37° C./5% CO₂ overnight to allow for celladherence. Prior to infection, the monolayers were washed three timeswith Dulbecco's phosphate buffered saline (DPBS).

Virus Preparation—The virus used for the assay was the influenza type Astrain A/Hong Kong/8/68. The virus was obtained from ATCC (catalog no.VR-544) and stock virus pools were produced in MDCK cells. A pretiteredaliquot of virus was removed from the freezer (−80° C.) and allowed tothaw slowly to room temperature in a biological safety cabinet. Viruswas resuspended and diluted into assay medium (DMEM supplemented with0.5% bovine serum albumin, 2 mmol/L L-glutamine, 100 U/mL penicillin,100 μg/mL streptomycin, 0.1 mM NEAAZ, 1 mM sodium pyruvate, and 1 μg/mLTPCK-treated trypsin) such that the amount of the virus added to eachwell in a volume of 100 μL was the amount determined to yield 90 to 95%cell killing at five days post-infection.

Plate Format—each plate contained cell control wells (cells only), viruscontrol wells (cells plus virus), drug toxicity wells (cells plus drugonly), drug colorimetric control wells (drug only) as well asexperimental wells (drug plus cells plus virus). Samples were tested intriplicate with eleven half-log dilutions per compound.

Anti-Bovine Diarrhea Virus (Surrogate Hepatitis C) Cytopropection Assay:

Cell preparation—MDBK cells (ATCC catalog no CCL-22) were passaged inT-75 flasks prior to use in the antiviral assay. On the day precedingthe assay, the cells were split 1:2 to assure they were in anexponential growth phase at the time of infection. Total cell andviability quantification was performed using a hemocytometer and TrypanBlue dye exclusion. Cell viability was greater than 95% for the cells tobe utilized in the assay. The cells were resuspended at 1×10⁵ cells permL in tissue culture medium (Dulbecco's Modified Eagles Medium, “DMEM,”supplemented with 10% horse serum, 2 mmol/L L-glutamine, 100 U/mLpenicillin, and 100 μg/mL streptomycin) to the drug-containingmicrotiter plates in a volume of 100 μL. The microtiter plates wereincubated at 37° C./5% CO₂ overnight to allow for cell adherence.

Virus Preparation—A pretitered amount of virus was removed from thefreezer (−80° C.) and allowed to thaw slowly to room temperature in abiological safety cabinet. Virus was resuspended and diluted into tissueculture medium such that the amount of virus added to each well in avolume of 100 μL was the amount determined to yield 90 to 95% cellkilling at five days post infection.

Plate Format—each plate contained cell control wells (cells only), viruscontrol wells (cells plus virus), drug toxicity wells (cells plus drugonly), drug colorimetric control wells (drug only) as well asexperimental wells (drug plus cells plus virus). Samples were tested intriplicate with eleven half-log dilutions per compound.

Efficacy and Toxicity XTT Used for Influenza and BVDV

Following incubation at 37° C. in a 5% CO₂ incubator, the test plateswere stained with the tetrazolium dye XTT(2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazoliumhydroxide) purchased from Sigma (St. Louis, Mo., catalog no. X4626).XTT-tetrazolium was metabolized by the mitochondrial enzymes ofmetabolically active cells to a soluble formazan product, allowing rapidquantitative analysis of the inhibition of virus-induced cell killing.XTT solution was prepared daily as a stock of 1 mg/mL in RPMI1640.Phenazine methosulfate (PMS; Sigma catalog no. P9625) solution wasprepared at 0.15 mg/mL in PBS and stored in the dark at −20° C. XTT/PMSstock was prepared immediately before use by adding 40 μL of PMS per mLof XTT solution. Fifty microliters of XTT/PMS was added to each well ofthe plate and the plate was reincubated for four hours at 37° C. Plateswere sealed with adhesive plate sealers shaken gently or invertedseveral times to mix the soluble formazan product and the plate was readspectrophotometrically at 450/650 nm with a Molecular Devices Vmax platereader.

Data Analysis—Raw data was collected from the Softmaz Pro software andimported into a Microsoft Excel XLfit4 spreadsheet for analysis by 4parameter curve fit calculations. Results in the form of EC₅₀, TC₅₀ andTI are provided in Table 1.

TABLE 1 In vitro Results of Tested CompoundsMDCK/Influenza_(A/Hong Kong/8/68) MDBK/BVDV_(NADL) EC₅₀ TC₅₀ EC₅₀ TC₅₀Compound (μM) (μM) TI (μM) (μM) TI Ribavirin 16.3 79.0 4.9 5.2 61.1 12.0Amantadine 0.1 >10.0 >90.9 Not Tested mPEG₃-N-Ribamidine67.8 >100.0 >1.5 >100.0 >100.0 — mPEG₆-N-Ribamidine >100.0 >100.0— >100.0 87.6 — mPEG₇-N-Ribamidine >100.0 >100.0 — >100.0 >100.0 —mPEG₅-N-Ribavirin (two lots) >100.0 >100.0 — >100.0 >100.0 —Di-mPEG₅-N-Ribavirin 5.5 19.5 3.5 >100.0 >100.0 —Di-mPEG₇-N-Ribavirin >100.0 >100.0 — >100.0 >100.0 — Ribavirin 18.2 50.02.8 8.1 69.8 8.7

Infection of MDCK cells with Influenza A virus is cytopathic andresulted in cell death. Treatment of infected cells with ribavirin wascytoprotective with an EC₅₀=18.2 μM. Influenza A virus-inducedcytotoxicity was also inhibited in the presence of mPEG₃-N-ribamidine(EC₅₀=67.8 μM). Similarly, di-mPEG₅-N-ribavirin was protective againstinfluenza A virus (EC₅₀=5.5 μM), although the test molecule was toxic toMDCK cells (TC₅₀=19.5 μM). Thus, the therapeutic index (TI) for thismolecule is 3.5.

Infection of MDBK cells with Bovine Viral Diarrhea virus (BVDV) is usedas a surrogate model for Hepatitis C viral infection. Ribavirinexhibited potent cytoprotection in BVDV-infected cells (EC₅₀=8.1 μM),however none of the PEG-conjugates tested was efficacious in this modelat concentrations up to 100 μM.

Based on the data obtained, further testing of the conjugates iswarranted.

What is claimed is:
 1. A method of treating influenza, comprisingadministering to a patient, a compound having the following structure:

wherein: R² is hydrogen or hydroxyl; R³ is hydroxyl when R² is hydrogenand R³ is hydrogen when R² is hydroxyl; (a) is two; X is an amidelinkage; and each POLY is independently a water-soluble, non-peptidicoligomer, and 5′-phosphates and 3′,5′-cyclic phosphates thereof andammonium and alkali metal salts of each of the 5′-phosphates and3′,5′-cyclic phosphates.
 2. The method of claim 1, wherein the weightaverage molecular weight of each water-soluble, non-peptidic oligomer isless than 400 Daltons.
 3. The method of claim 1, wherein eachwater-soluble, non-peptidic oligomer is a poly(alkylene oxide).
 4. Themethod of claim 3, wherein each poly(alkylene oxide) is a poly(ethyleneoxide).
 5. The method of claim 4, wherein the poly(ethylene oxide)oligomer has a number of repeating monomers in the range of from 1 to30.
 6. The method of claim 5, wherein the water-soluble, non-peptidicoligomer has a number of repeating monomers in the range of from 1 to10.
 7. The method of claim 3, wherein the poly(alkylene oxide) includesan alkoxy or hydroxy end-capping moiety.
 8. The method of claim 1,wherein the compound is administered as part of a composition comprising(i) the compound of claim 1, and (ii) a pharmaceutically acceptableexcipient.
 9. The method of claim 1, wherein a dosage form comprisingthe compound of claim 1 is administered.
 10. The method of claim 1,wherein the compound is administered orally.
 11. The method of claim 8,wherein the composition is administered orally.
 12. The method of claim1, wherein the amount of compound administered is in the range of 0.01mg to 750 mg.
 13. The method of claim 1, wherein the compound isselected from a compound of the formula

wherein for each occurrence, n is an integer from 1 to 15; and5′-phosphates and 3′,5′-cyclic phosphates thereof and ammonium andalkali metal salts of each of the 5′-phosphates and 3′,5′-cyclicphosphates.
 14. The method of claim 13, wherein for each occurrence, nis an integer from 1 to
 10. 15. The method of claim 14, wherein for eachoccurrence, n is
 5. 16. The method of claim 13, wherein the compound isadministered as part of a composition comprising (i) the compound ofclaim 13, and (ii) a pharmaceutically acceptable excipient.
 17. Themethod of claim 13, wherein the compound is administered orally.
 18. Themethod of claim 16, wherein the compound is administered orally.
 19. Themethod of claim 13, wherein the amount of compound administered is inthe range of 0.01 mg to 750 mg.